Mewtwo made its first appearance in Tauros the Tyrant, where Red spots its still-unfinished body floating inside a tank. Red overhears Team Rocket scientist Blaine talking about how there was not enough genetic material in Mew's cell to complete the body. Later, Mewtwo is eventually finished, but it turns on its creators and escapes.
Mewtwo then proceeded to go on a rampage in Cerulean City, where it would be nicknamed the "Monster of Cerulean City." Red decided to pursue Mewtwo after learning of its powers and found it inside the Cerulean Cave. Upon entering, Mewtwo attacks Red with a tornado made of psychic energy. Red is saved by Blaine, who reveals that he was a member of Team Rocket and created Mewtwo. Blaine reveals that because he did not have enough of Mew's cells, he was forced to use his own cells to finish Mewtwo's body. However, a few of Mewtwo's cells found their way into his own arm, which gave the two the ability to sense one another. Despite this ability, Blaine stated that he would eventually die from Mewtwo's cells taking over his body.
Blaine attempts to sacrifice himself by charging straight at Mewtwo in an attempt to kill it, but they both survive the attack. Angered from the attack, Mewtwo begins going on the offensive, forcing Red to try and defend himself. The battle ends when Red sends Pika hurtling down Mewtwo's Psywave twister with a Master Ball inside his mouth. With Mewtwo captured, Blaine notices that the pain in his arm subsided, which meant that Mewtwo was willing to start over with its creator.
In the Yellow chapter, Mewtwo makes its first appearance in Purrrr-sian, floating inside a tank, asleep. When Yellow visits Cinnabar Island, she encounters Blaine and Mewtwo training together for their upcoming battle against the villainous Elite Four. At Cerise Island, Mewtwo teamed up with Blaine and Yellow to battle the leader of the Elite Four, Lance. Despite Mewtwo's power, Lance's Pokémon proved to be equally as powerful as it. During the battle, Blaine reveals that Mewtwo is unable to fight very far from Blaine or else it will put a lot of strain on their bodies. In addition to this, Mewtwo can only remain outside of its Master Ball for three minutes before it has to return.
Having no choice, Blaine has Mewtwo target Lance's Ultra Balls so that he cannot release the rest of his Pokémon. Mewtwo manages to destroy the Ultra Balls' releasing mechanism, but Lance reveals that his other Pokémon were outside the entire time, making the attack useless. The attack proves to be too much for Blaine and he collapses. Despite this, Mewtwo continues to attack Lance with the intention of putting its life on the line. Lance manages to stop Mewtwo when he points out that by continuing to battle, Blaine will eventually die from the strain as well. Not willing to kill its Trainer, Mewtwo forcefully sends itself back into its Master Ball.
In the Gold, Silver & Crystal chapter, Mewtwo is left in Blaine's laboratory while Blaine participates in the Pokémon League's opening ceremony with the other Gym Leaders of Kanto and Johto. When Blaine meets up with the legendary Entei and becomes its partner, Blaine asks it to use its healing flames to heal his arm and sever the link he has with Mewtwo. Once Entei does so, Mewtwo immediately leaves Cinnabar Island, now a free Pokémon.
During the FireRed & LeafGreen chapter, Mewtwo makes a reappearance as an ally. It first reappears in It Takes Patience, Knowledge and a Really Quick Beedrill, where it reveals that it had rescued the injured Lorelei after her defeat at the hands of Sird. Although Red is initially confused as to why Mewtwo had shown up, Mewtwo reveals that it wishes to fight with Red against Team Rocket. Mewtwo reveals that after leaving its master—Blaine—it has gained the ability to speak through telepathy, but only to those it trusts, such as Yellow, Red, and Blaine. Red accepts Mewtwo as his ally, much to the shock of Blue and Green.
They travel to Trainer Tower, where Team Rocket's base of operations is located. Although a group of Unown under Sird's control attempt to attack and stop the group, Mewtwo easily swats them away with its powerful Psywave technique. Upon landing on the Trainer Tower's roof, Blue and Green are fooled by holograms of their kidnapped loved ones and are grabbed by mechanical arms when they attempt to save them. Mewtwo manages to save the two with its spoon and takes the three down to another level of the building. Upon arriving, Mewtwo uses its abilities to help locate Professor Oak and Green's parents.
The group is soon attacked by Giovanni who has an army of Deoxys clones—called the Deoxys Duplicates—under his command. While the others face the clones, Mewtwo goes to directly attack Giovanni. Mewtwo uses telepathy to communicate with Giovanni, but only because it wants Giovanni to feel its wrath. Mewtwo attempts to attack, but the Deoxys Duplicates step in to defend Giovanni. Mewtwo tells Giovanni that it refuses to let Team Rocket use Deoxys for their evil plans like they almost did with itself.
Mewtwo trapped by the M2 Bind
When Giovanni explains Deoxys's origins and how it's under his complete control, Mewtwo becomes enraged. Noting how powerful Mewtwo can be, Giovanni has the Trainer Tower's computer, R, cover Mewtwo in a specialized armor. Called the "M2 Bind", the armor is designed to restrict its powers. With the M2 Bind surrounding its body, Mewtwo is unable to move while Giovanni makes his escape. Eventually, Mewtwo comes up with a plan and tells Red, Blue, and Green have their Pokémon fire off Frenzy Plant, Blast Burn, and Hydro Cannon, at the exact same time from different directions. The moves' types cancel each other out before hitting Mewtwo, thus eliminating any pain Mewtwo would feel. Now free, Mewtwo takes the chance to destroy R by cleaving the Trainer Tower in two with its spoon.
With Blue and Green too tired to continue, Mewtwo leaves with Red to chase after Giovanni. They arrive at the Team Rocket airship, which has turned into a stadium. Red proposes a challenge with him and Mewtwo against Giovanni and Deoxys. After agreeing, the two Pokémon begin a fierce battle. Although seemingly evenly matched, Deoxys's ability to change its Formes gives it a major advantage over Mewtwo. Eventually, the battle is decided when Mewtwo fires an attack directly at Deoxys's core, severely injuring it and rendering it unable to battle.
Despite this victory, the airship ages out of control when Carr goes crazy and announces his plan to crash it into Vermilion City and blow it up with his ten Forretress. To protect his friends, Red tells the newly-befriended Deoxys to send Mewtwo and everyone else on board the airship to safety via a black hole. Eventually, Mewtwo, with an unconscious Yellow in its arms, lands near Vermilion City. After the airship lands safely, Mewtwo is the only one to notice that it was actually Mew who stopped it from crashing. Realizing that since it was cloned from Mew's eyelash, it now has a something to chase its roots back to and thanks Deoxys for the sharing the same sentiments, as well as the battle they had.
After Deoxys leaves, a severely-injured Sird returns with the intent of recapturing Deoxys. She grabs a Poké Ball and shoots a beam at Deoxys, but Mewtwo, Red, Blue, Green, and Silver jump in the way of it. Although the five Dex-holders are turned to stone, Mewtwo was not.
What had happened to Mewtwo at the climax of the FireRed & LeafGreen chapter was unknown until Sird's reappearance at the end of this chapter. She reveals that the reason for the Pokédex holders' petrifaction was because Mewtwo had clashed with the Pokémon she was intending to use on Deoxys, Darkrai. Mewtwo was revealed to have continued chasing Sird after the Pokédex holders' petrifaction, but eventually lost her.
After a long absence, it is revealed that Mewtwo had become the protector of the Pokémon Village. When Team Flare arrived and began abducting the Pokémon at the village, Mewtwo confronted them in battle. When X and Blue arrive at the village to stop Team Flare once and for all, they find Mewtwo in battle with Lysandre, who manipulates Mewtwo into battling X and Blue so that it may be weakened enough to be captured. Despite having battled together, Blue's attempt to get Mewtwo to recognize him ends in failure.
During the battle, Mewtwo attacks X and Blue, but redirects the attack towards Lysandre to hit him instead. Mewtwo then proceeds to Mega Evolve into Mega Mewtwo Y. As Mewtwo battles Lysandre's Pokémon, Blue notices another person in the distance who was responsible for Mega Evolving Mewtwo.