When the player first rides the ship to Vermilion City, a man will bump into them as they board and explain that he lost his granddaughter. When the player first enters the lower deck, a crewman will deny them access to the rest of the ship, asking them to locate a co-worker who is slacking off on the floor above. The crewman is a Sailor in a previously empty room (in Generation IV, the Sailor is found sleeping in a cabin before the player is asked to find him), and once he is defeated in a Pokémon battle, the crewman below will let the player pass. After traversing through the lower deck, the player will find the man's granddaughter trying to get the captain to play with her. The player takes her back to her grandfather, at which point the S.S. Aqua will arrive in Vermilion City and the player can disembark.
After the initial trip to Kanto (which can happen on any day of the week), the S.S. Aqua can be sailed from Kanto to Johto every Sunday and Wednesday and from Johto to Kanto each Monday and Friday. Trips after the initial one will end when the player sleeps in the bed in their cabin, which heals their party.
Trainers can also be encountered and battled on the ship each voyage. In Pokémon Gold, Silver, and Crystal, if the player defeats one or more Trainers on the ship and then boards it again for the same voyage (even on the same day), the Trainers can be battled and defeated again, and the player can keep re-boarding and re-battling the Trainers an unlimited number of times. However, in the Generation IV remakes, once an individual Trainer is defeated, that Trainer can never be battled again, regardless of when or how often the player re-boards the S.S. Aqua.
The S.S. Aqua first appeared in Off Course with Corsola, where Lt. Surge unintentionally brought Crystal to the ship from Union Cave through a secret underwater tunnel. Soon after, the ship landed in Olivine City, which Crystal, having been in Union Cave to get to the western side of Johto, was pleased to reach. It was also revealed that Lt. Surge tended to use the ship for personal purposes.
The ship made a brief cameo appearance in All About Arceus III as one of the locations where Silver and his Weavile discovered a Plate, receiving it from the ship's captain.
In both the Generation II games and Pokémon HeartGold and SoulSilver, if the player goes to sleep, wakes up, and hears the announcement about docking, checking the Pokégear map will still show the player in the ocean between Johto and Kanto. This is because "S.S. Aqua" is actually a location on the map, and thus cannot change.