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Playable characters
- Ash Ketchum - The main protagonist of the game, and the only character playable in single player. His Pokémon are Pikachu, Bulbasaur, and Squirtle.
- Gary Oak - Ash's rival, and Professor Oak's grandson. His Pokémon are Krabby, Growlithe, and Nidoran♀.
- Brock - One of Ash's traveling partners, and Pewter City's Gym Leader, specializing in Rock-type Pokémon. His Pokémon are Vulpix, Geodude, and Zubat.
- Misty - One of Ash's traveling partners, and Cerulean City's Gym Leader, specializing in Water-type Pokémon. Her Pokémon are Horsea, Psyduck, and Staryu.
- Lt. Surge - Vermilion City's Gym Leader, specializing in Electric-type Pokémon. His Pokémon are Jolteon, Raichu, and Magneton.
- Erika - Celadon City's Gym Leader, specializing in Grass-type Pokémon. Her Pokémon are Tangela, Weepinbell, and Gloom.
- Koga - Fuchsia City's Gym Leader, specializing in Poison-type Pokémon. His Pokémon are Venomoth, Voltorb, and Golbat.
- Sabrina - Saffron City's Gym Leader, specializing in Psychic-type Pokémon. Her Pokémon are Abra, Hypno, and Alakazam.
- Blaine - Cinnabar Island's Gym Leader, specializing in Fire-type Pokémon. His Pokémon are Arcanine, Charmeleon, and Magmar.
- Tracey Sketchit - Traveled with Ash and Misty through the Orange Islands after Brock chose to stay behind. His Pokémon are Marill, Venonat, and Scyther.
- Team Rocket (Jessie, James, and Meowth) - A trio of Pokémon thieves who constantly follow Ash and co. around with the intent of stealing his Pikachu. Their Pokémon are Weezing, Arbok, and Golbat.
- Giovanni (unlockable) - The boss of Team Rocket, and Viridian City's Gym Leader, specializing in Ground-type Pokémon. His Pokémon are Sandslash, Persian, and Nidoking.
- Ritchie (unlockable) - The trainer whom Ash lost to in the Indigo League tournament. His Pokémon are "Sparky" (Pikachu), "Zippo" (Charmander), and "Happy" (Butterfree).
- Lorelei (unlockable) - A member of the Indigo League's Elite Four, who specializes in Ice-type Pokémon. Her Pokémon are Cloyster, Dewgong, and Poliwhirl.
- Bruno (unlockable) - A member of the Indigo League's Elite Four, who specializes in Fighting-type Pokémon. His Pokémon are Onix, Hitmonchan, and Primeape.
- Mewtwo (unlockable via button code; can only be controlled by Player 2) - A Pokémon created by Team Rocket by cloning a Mew. Its' Pokémon are clones of Pikachu, Bulbasaur, and Squirtle.
NPCs
- Professor Samuel Oak - The leading authority of Pokémon research. He serves as the tutor for teaching you basic gameplay and techniques at his lab.
- Gary Oak with evolved Pokémon - As the final stage before battling Mewtwo, Gary battles you a second time.
- Cassidy and Butch - Rivals of Jessie and James, respectively, who work for Team Rocket. The two of them battle you in the Spa Service as a bonus challenge.
- Nurse Joy - A Pokémon nurse who has identical-looking female relatives (all are nurses with same name). She is seen at the Pokémon Center to modify the game settings.
- Officer Jenny - A policewoman who has identical-looking female relatives (all are all policewomen with same name). She is seen at the Pokémon Center to modify the game settings.
- Delia Ketchum - Ash's loving mother. She can be seen and heard in cutscenes after clearing the 1P Stadium at any difficulty level.
- Jigglypuff - A Pokémon with a desire to sing. It is seen or heard at all the modes except 1P Stadium and 2P Stadium.
- Togepi - Misty's Pokémon. It can be seen and heard when playing Super Easy Mode in the Mimic Mansion, Time Zone, Marathon, and in cutscenes after clearing the 1P Stadium at any difficulty level.