In the 2012 TV series, Retro-Mutagen is an antidote to the effects of the regular Mutagen. The substance was invented by Donatello through a long period of trial and error. A much more potent form of retro-mutagen was accidentally formulated by Michelangelo from Donnie's existing retro-mutagen samples.
Retro-mutagen is made from regular mutagen, but the source mutagen must undergo complex chemical alterations that involve April O'Neil's DNA. April was herself born a Human-Kraang hybrid mutant, inheriting traits from her mother whose family line had been genetically altered by Kraang experiments. One of April's special properties is an immunity to mutagen itself, which is instrumental to how Donnie's retro-mutagen reverses the effects of mutation.
Unlike regular Mutagen which is cyan in color, Donatello's successful batches of Retro-Mutagen are orange in color.
2012 TV series
- Mutagen Man Unleashed - Donnie creates a batch of unsuccessful retro-mutagen that freezes mutagen on contact. Donnie would later reluctantly use it against Timothy who had gone insane after repeatedly siphoning mutagen into his tank.
- Mikey Gets Shellacne - Michelangelo gets into another reject batch of retro-mutagen, sees the label "Super cooled", and misconstrues it as a promise that using it will make him "super cool". But using the substance gives Mikey shell-acne which is actually the first signs of a reaction that would cause him to explode if untreated. Donnie used a much-sought-after molecular centrifuge in creating the antidote.
- The Lonely Mutation of Baxter Stockman - After a long time trying, Donnie created the antidote so he could cure Kirby O'Neil and Splinter. Baxter and Rahzar also wanted it to cure themselves, but in the end the turtles won and cured Kirby, although the other sample of retro-mutagen Donatello had created was destroyed. Splinter decided to accept his fate as a mutant.
- Return to New York - Baxter mentions he's experimenting with arthropod DNA to develop a cure for Karai's mutation.
- Serpent Hunt - Donatello reveals he's been working on a new batch of retro-mutagen in the last months.
- The Pig and The Rhino - Donnie had made three spray canisters of retro-mutagen in hopes of curing Karai. He gave one to himself, Leo and Raph while splitting up to locate Karai. Bebop and Rocksteady learned about this and wanted to cure themselves of their mutations, but got into a fight with the Turtles to see who gets it first. When Leo sprayed retro-mutagen on Karai, it has no effect due to her unique and special mutation according to Donnie.
- Battle for New York, Part 1 - Michelangelo accidentally created a super retro-mutagen formula that can transform huge amounts of mutagen into retro-mutagen. The Turtles stole a Technodrome and used its mutagen tank to un-mutate the entire population of New York, including Kirby.
- The Creeping Doom - Donatello used it to make a weed killer to defeat Creepweed.
- Owari - Donatello puts retro-mutagen into empty light bulbs, using them as containers, with the intention of reverting Super Shredder back to his human form. Michelangelo throws one of the retro-mutagen filled light bulbs at Stockman-Fly, turning him back to normal. Donatello throws another one of the retro-mutagen light bulbs at Super Shredder, but it has no effect.
- Donatello has stated each dose of retro-mutagen requires ten canisters of mutagen to synthesize properly.
- April's unique DNA is the key ingredient, due to her apparent immunity to mutagen.
- A similar serum was created in the 1987 series by Donatello in Splinter No More to return Splinter to his human form. Unlike the Retro-Mutagen, the effects were temporary.
- When Donatello asks Michelangelo what did he use to create the super retro-mutagen, Mikey made up a few components' names, but the first one called Uranate is an actual substance.
- The other components he said he used were Anolite, moronic acid, and some garlic cappuccino pizza.