After made a heist in Gotham Palace and tied the public along with Riddler, Penguin and Catwoman, the Joker used his Jokermobile to reach the chemical factory and stole the Duplicator Ray. Batman and Robin tried to stop him and his minions, but the Joker escaped along with the other villains to a frozen food factory, and once there, he tried to cook Batman and Robin with a huge oven, after Catwoman tried to make Batman a bad guy, without apparent success. But the Dynamic Duo escaped.
After leaving the factory, the Joker and the other villains used a U.S. rocket to reach a space station built by Russia and United States. However, along Riddler and Penguin, the Joker concluded that Catwoman could betray them, because she was in love with Batman. For that reason, along with the other villains, the Joker locked Catwoman in a special room and launched her into space to kill her. However, Batman and Robin saved Catwoman from her planned death; and faced Riddler, the Penguin and the Joker in the main room of the station. After an antigravity fight, Batman defeated the villains, including Joker, leaving them unconscious, and they were taken into custody offscreen after this.
However, in reality, the Joker, along with the Riddler and the Penguin, who had doubled themselves with the Duplicator Ray, and Batman had send to prison their duplicates. After Batman returned to his normal state, he reached to this conclusion. However, during this time, the Joker, the Riddler and the Penguin robbed important artworks from Gotham Museum, one more higher than the previous. But Batman, Robin and Catwoman appeared on the scene to stop the villains, whereby the Joker aboard the Penguin's Blimp, along the Riddler and a few henchmen from his gang. However, Batman, Robin and Catwoman defeated his henchmen along the Riddler and the Penguin. Batman and Robin tried to show the Joker that he hasn't a chance for escape this time. However, Joker threw a fart for neutralize the Dynamic Duo and fell into the Big Top of Gotham Circus. However, the artists of the circus defeated him with slapthicks and locked him inside a van full of police clowns, making mourn the Joker. He was never seen in the movie after this, but it's likely that after that, he was taken into custody for his crimes.
- Although this version of the Joker is based on the incarnation of the 1960's Batman TV show and the 1966 film of the same name, that was portrayed by the late Cesar Romero, he doesn't have the makeup covered moustache of the late actor, though that in several representations of the Romero's incarnation (like toys and merchandise), the makeup covered moustache was showed sligthly, like in the series.
- In the trailer of Batman vs. Two-Face, he was clearly seen with the Penguin and the Riddler as they have returned from Batman: Return of the Caped Crusaders. It was unknown if they will have any lines because Two-Face is the main antagonist for the sequel. Other characters like Egghead, Mr. Freeze, Shame and Clock King also appeared in the first movie as they were non-speaking characters and they return for the sequel too.