Two experiments were performed to characterize the process of postnatal demasculinization in Japanese quail. In the first experiment, it was shown that estradiol (E2) can complete female demasculinization during the first 4 weeks of life. By contrast, E2 did not demasculinize sexual behavior and cloacal gland in neonatally castrated males. Neonatally gonadectomized females preferentially performed mount attempts when tested in their home cage by comparison to a test arena. In Experiment 2, E2 Silastic implants (40-mm) maintained full copulatory behavior in castrated males but not in females. This large dose of E2 did not demasculinize adult sexually active birds (males or females) even if treatment lasted for 1 month. It is concluded that E2 can demasculinize sexual behavior only in females and only if treatment is performed in very young birds.