Although the question of to whom a male directs his mating attempts1,2 is a critical one in social interactions, little is known about the molecular and cellular mechanisms controlling mammalian sexual preference. Here we report that the neurotransmitter 5-hydroxytryptamine (5-HT) is required for male sexual preference. Wild-type male mice preferred females over males, but males lacking central serotonergic neurons lost sexual preference although they were not generally defective in olfaction or in pheromone sensing. A role for 5-HT was demonstrated by the phenotype of mice lacking tryptophan hydroxylase 2 (Tph2), which is required for the first step of 5-HT synthesis in the brain. Thirty-five minutes after the injection of the intermediate 5-hydroxytryptophan (5-HTP), which circumvented Tph2 to restore 5-HT to the wild-type level, adult Tph2 knockout mice also preferred females over males. These results indicate that 5-HT and serotonergic neurons in the adult brain regulate mammalian sexual preference.