Previous studies have shown that adult male rats, in which brain estrogen formation was inhibited neonatally by SC administration of the aromatase inhibitor 1,4, 6-androstatriene-3,17-dione (ATD), show an altered sexual partner preference. When tested in a three-compartment box, such gonadally intact ATD males approach and mate both with the estrous female and the sexually active male, whereas normal males prefer to approach and mate with the estrous female, avoiding the stimulus male. After castration in adulthood and estradiol treatment, ATD males prefer sexually active males. Similarly treated normal males prefer estrous females, and estrous females prefer to mate with males. In the present study, we asked what stimulus characteristics of active males vs. estrous females determined the different sexual preferences of males, ATD males, and of females. Were they chemosensory cues or more distal cues such as actually seeing and hearing the stimulus animals or the reward of sexual activity with the stimulus animals? Sex differences in preference were evident when animals were given a choice between soiled bedding from estrous females and from sexually active males. ATD and control males spent significantly more time on soiled bedding from estrous females than on soiled bedding from sexually active males. Control females spent significantly more time on soiled bedding from sexually active males than on soiled bedding from estrous females. More distal cues, such as seeing and hearing the stimulus animals, revealed differences in preference between control males and females, but not between ATD and control males. Physical interaction with the stimulus animals was a prerequisite for revealing differences in preference between ATD and control males. Then, the behavior of ATD males was clearly intermediate between that of normal male and female rats. In conclusion, neonatal estradiol is important for the psychosexual development of the male rat. However, the present data suggest that the psychosexual development of the male rat also requires either prenatal estradiol or perinatal testosterone.