Repeated testing for masculine sexual behavior influences female sex preference in males. Males perinatally treated with aromatase inhibitors show male preference, but also copulate with the receptive female. Such copulation modifies sex preference most likely because of its rewarding properties. In this study, we intended to equal the incentive value of both stimuli -in the sex preference test- by using receptive females with vaginal occlusion. Vehicle and letrozole-treated (0.56 μg/kg, gestation days 10-21) males were repeatedly tested for sex preference at 40, 55, 70, 85 and 100 days of age. These ages were selected because males of 40 days are unable to copulate, while by 100 days of age almost all males show the complete repertoire of masculine sexual behavior. At 40 days of age, males of all groups fail to show sex preference and none of them was able to copulate. In controls of 100 days of age all males showed female-sex preference and all intromitted the female. A large proportion (44%) of vehicle-treated males that could not copulate the female showed male preference. Twenty to 30% of the prenatally letrozole treated males also had same-sex preference even if they could copulate; and most of them (67%) had a male preference when copulation was precluded. These data support the idea that copulation is crucial for developing a female preference in control animals. The results suggest that brain changes produced by estrogens along early development and stimuli coming from the partner are essential for shaping sex preference.