During sexual differentiation males and females are exposed to different levels of testosterone, which promotes sex differences in the adult brain and in behavior. Testosterone can act after aromatization or reduction via a number of steroid hormone receptors. Here we provide new evidence that the androgen receptor (AR) is essential for sexual differentiation in mice. We used mice carrying the testicular feminization (Tfm) mutation of the AR. Adult Tfm males, wild-type male and female littermates were gonadectomized and given subcutaneous estradiol implants. In all sexually dimorphic traits, Tfm males had responses equivalent to females and different from males. In simultaneous choice tests, males spent significantly more time investigating female-soiled bedding, whereas females and Tfm males preferred to investigate male-soiled bedding. Tfm males and females did not have a partner preference in tests with awake stimulus animals, whereas males showed a preference for females over males. Exposure to male-soiled, but not clean, bedding produced a significant increase in c-Fos-immunoreactive cells in the medial preoptic area and bed nucleus of the stria terminalis in Tfm males and females, no increase was noted in males. Masculine sexual behavior (mounting and thrusting) was not sexually dimorphic, and all groups displayed these behaviors. Our results support data collected in humans suggesting a role for the androgen receptor in sexual differentiation of social preferences and neural responses to pheromones.