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Like all other Pokémon RPGs, Diamond and Pearl begin in the protagonist's hometown. After viewing a television report about a media-conducted search for a Red Gyarados, which was spotted at a faraway lake (Johto's Lake of Rage) the protagonist and his or her best friend travel together to check the local lake for a Pokémon like it. They spot Professor Rowan, a Pokémon evolution researcher, and his assistant, the playable character not selected in the game: Lucas (boy) or Dawn (girl). After a short discussion, the professor and his assistant leave the lake, leaving a briefcase behind. When they are attacked by wild Starly, the protagonist and his or her rival examine the case. The player is then given a choice among the three Pokémon found in the briefcase (Turtwig, Chimchar, or Piplup) with which to battle the Starly. After defeating the Starly, Lucas or Dawn retrieves and returns the briefcase to the professor. Noticing that a bond has been forged between the young protagonist and his or her chosen Pokémon, Rowan offers it to him or her, asking that he or she embark on a journey and fill his or her Pokédex.

The protagonist encounters the main antagonist, Team Galactic, early in the game, when he or she must save Professor Rowan from its thugs; however, its motives are unclear until later. The protagonist encounters the Team twice (when it takes over a wind farm and when it sets up a base in Eterna City) before it takes over Sinnoh's three lakes in an attempt to capture the Mirage Pokémon (Uxie, Azelf, and Mesprit). Shortly after the player earns his or her seventh Gym Badge, Team Galactic captures the Mirage Pokémon and imprisons them inside the science laboratory of the Team Galactic Headquarters Building, where its members extract crystals from the Pokémon to create the Red Chain, an object that can control the legendary Pokémon Palkia (in Pearl) or Dialga (in Diamond) (both in Pokémon Platinum, though Giratina appears after this). After releasing the trio, the protagonist is able to access the cave atop Mt. Coronet, where the leader of Team Galactic awakens Dialga or Palkia. The legendary Pokémon's powers begin to overwhelm Sinnoh, causing the newly free Uxie, Azelf, and Mesprit to attempt to stop it. The player then battles Palkia/Dialga; after defeating or capturing the Pokémon, Sinnoh returns to normal. After this, the player will continue, eventually battling the Sinnoh Region Pokémon League's Elite Four: Aaron, Bertha, Flint, and Lucian. After defeating all four members, the player will battle the Sinnoh League champion, a woman named Cynthia, who had appeared before in the game. If the protagonist beats Cynthia, he or she is the new Sinnoh League champion, beating the game. After the player beats the game, there is a new island to explore that is filled with other types of Pokémon. His or her old friend, who challenged him or her to battles multiple times before, will be waiting for him or her here. The old friend will challenge the player to another battle. On this island, there are also stores and a tournament center.