Harley Quinn is the frequent accomplice and lover of the Joker, whom she met while working as a psychologist at Gotham City's Arkham Asylum, where the Joker was a patient. Her name is a play on the name "Harlequin", a character which originated in the commedia dell'arte. The character has occasionally been depicted as an antihero, and she has teamed up with fellow villains Catwoman and Poison Ivy, the latter frequently serving as Quinn's close friend and recurring ally. Harley Quinn has also been depicted as a member of the Suicide Squad, a team of villains and antiheroes alike who carry out secret missions.

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The character was originally voiced by Arleen Sorkin in various tie-ins to the DC animated universe. Since then, she has also been voiced by Hynden Walch and Tara Strong in either DC Animated Showcases or in various video games. In the Birds of Prey television series, she was portrayed by actress Mia Sara. The character made her live-action cinematic debut in the 2016 film Suicide Squad, portrayed by Margot Robbie.

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Harley Quinn first appeared in the Batman: The Animated Series episode "Joker's Favor", as what was originally supposed to be the animated equivalent of a walk-on role; a number of police officers were to be taken hostage by someone jumping out of a pop out cake, and it was decided that to have the Joker do so himself would be too bizarre, although he ended up doing it anyway. Dini thus created a female sidekick for the Joker, who would become his love interest. Arleen Sorkin, a former star of the soap opera Days of Our Lives, appeared in a dream sequence on that series in which she wore a jester costume; Dini used this scene as an inspiration for Quinn. Having been friends with Sorkin since college, he incorporated aspects of her personality into the character. Quinn was also inspired by a mutual female friend's "stormy (but nonviolent) relationship", according to Timm.

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The 1994 graphic novel The Batman Adventures: Mad Love recounts the character's origin story. Written and drawn by Dini and Timm, the comic book is told in the style and continuity of Batman: The Animated Series. It describes Dr. Harleen Frances Quinzel, PhD as an Arkham Asylum psychologist who falls in love with the Joker and becomes his accomplice and on-again, off-again girlfriend. The story received wide praise and won the Eisner and Harvey Awards for Best Single Issue Comic of the Year. The New Batman Adventures series adapted Mad Love as the episode of the same name in 1999. It was the second "animated style" comic book adapted for the series, with the other being "Holiday Knights".

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Harleen becomes fascinated with the Joker while working at Arkham Asylum and volunteers to help treat him. She falls hopelessly in love with the Joker during their sessions, and she helps him escape from the asylum more than once. When Batman returns a badly injured Joker to Arkham, Harleen dons a jester costume to become Harley Quinn, the Joker's sidekick. The Joker frequently insults, ignores, hurts and even tries to kill Harley, but she always comes back to him, convinced that he truly loves her.