In the Rowdyruff Boys' self-titled debut episode, Mojo Jojo created them during a stay in prison. Desperate to find a way to create a power to equal to the Powerpuff Girls, Mojo Jojo placed an anonymous phone call to Professor Utonium, who was all too willing to tell "the stranger on the phone from Townsville Community College" his recipe for the girls' creation.

Finding the ingredients of sugar, spice, and everything nice "too girlish" for his needs, Mojo wanted something "tougher" and "more manly". So he made a few variations on the recipe with everything he needed available in prison: snips (underarm hair), snails (escargot from the prison cafeteria), a puppy dog tail (pulled from the Talking Dog), and murky black slime on the prison toilet (which was considered by Mojo to be Chemical X)). As with the Powerpuff Girls, the ingredients used to make the Rowdyruff Boys are based on the Nursery rhyme "What are Little Boys Made of?"

When the Powerpuff Girls and the Rowdyruff Boys first met, they nearly destroyed all of Townsville in the ensuing fight, which ultimately concluded with the Rowdyruff's leaving the girls bruised, broken, and beaten. As the girls recovered from the tears of the townsville citizens, they were so ashamed of their defeat they considered leaving, but Miss Bellum suggested a different way of fighting the boys, as they are naturally superior in combat. Taking her suggestion to the field, the Powerpuff Girls managed to destroy the Rowdyruff Boys with a single kiss on the cheeks, each to their counterpart. Being 10x irregular from the average boy, the kisses had 10x irregular the effect of disgust on the boys, which triggered an explosive reaction playing off of every little boy's greatest fear: cooties/kisses from a girl, which is claimed to be able to spread.