As an Antagonist
In the episode Nano, Nano was created as part of a government project by Dr. Marion Richards. Nano quickly developed intelligence and a separate, albeit initially immature and childish personality. However, as it had the intelligence of a three-year old, it could only make simplistic robots. Richards thought to update the bots after a coffee, as it reassembles a body with a drill to escape. Looking for a family Nano fled to the streets of New York. It quickly ran into Harry Parker. Using Harry's broken toys, the nanobots created a body and adopted Harry as its father. Harry decided to use it to break into stores but had a problem understanding what emotion Nano was having with its current body. During one break-in, Nano picked out traditional theatre masks of comedy and tragedy to show his emotions and facial expressions.
When breaking into 2nd Time Around, they met Casey Jones, who Nano knocked out, while the Turtles fought it off and also realized that Nano was more a confused child than a professional criminal. Discovering what Nano was, they decided that he was a threat and must be destroyed. During a fight at a junkyard, they caught it using an electromagnet and dumped it into a blast furnace. The police show up and arrest Harry.
However later in the episode Modern Love - The Return of Nano, a few parts of Nano survived and managed to completely regenerate over time. It watched a video of a family soap opera for a happy life and family, which then turned to Harry's transfer to prison.
Nano also wanting to build a happy family life broke Harry out of the prison that he was being held at. Nano then went back to the lab where he was created and kidnapped Dr. Marion Richards and seeing both of them as his parents, built a house in the lab wanting to have a "family", reenacting the video. But both began arguing which angered Nano, who wanted to have a happy family. Then Nano went to Coney Island where April O'Neil and Casey Jones were repairing the Ferris wheel. It brought gold, hoping to make them happy but they wished to go free.
This angered Nano, who was determined to keep his "family" together. The Turtles show up and battle it as amalgamates itself with more parts. Donatello and April modified a roller coaster's track into generating an electromagnetic pulse to defeat Nano.
Road to Heroism
In the episode Membership Drive, while salvaging Triceraton tech for Bishop, Stockman stumbled upon the nanobots remains attached to a burnt-out Triceraton ship. In order to put them under his command, Stockman made a microchip and placed it on one of the nanobots, which in turn sent its programming to the other nanobots. However, Stockman's over-ride programming reactivated Nano's personality and caused an internal conflict, alternately driving him between committing violence and trying to seek help. Nano broke out of the container which he was held in, built a new body using Triceraton tech, as well as making "Henchmen" from Bishop's lab, and escaped into the streets of New York.
While moving down the streets, Nano saw the Justice Force on TV and attempted to seek their help, but his scrambled judgement made him attack the team instead. Leonardo and Mikey, who were present at the Justice Force's headquarters to ask for their allegiance against the threat of the Tengu Shredder, recognized Nano, and after Ananda managed to analyze Nano's problem, they took the shrinking belt of Moleculo, who had come to apply for membership in the Justice Force, went inside Nano, and managed to remove the infected nanite from the rest of the system. Nano regained control over himself, formed a new body, and with some helpful introduction by Mikey, Leo and Ananda, became the newest member of the Justice Force, completing their membership drive. Having evolved his programming since his past encounters with the Turtles, he thanks them for their help, and apologizes for his "Lack of maturity".
Nano and the rest of the Justice Force were later on part of the Turtles' strike team against the Tengu Shredder.
Back to the Sewer
Nano has been confirmed to be in TMNT: Back to the Sewer as part of the Justice Force. He appears in the episodes Super Power Struggle and Wedding Bells and Bytes.