The injection before Day 12 of incubation of estradiol benzoate (EB) into Japanese quail eggs produces a complete behavioral demasculinization of adult males that will hatch from these eggs. These males never show copulatory behavior even after administration of high levels of exogenous testosterone (T). It is usually assumed that such a demasculinization normally takes place in female embryos under the influence of endogenous estrogens but few experimental data are available to confirm the validity of this model. A series of four experiments was performed during which R76713, a triazole derivative that specifically inhibits aromatase (estrogen synthetase) activity, was injected into quail eggs at different stages of incubation to prevent the production of endogenous estrogens. The consequences of these embryonic treatments on the T-activated sexual behavior in adults were then quantified. When injected before Day 12 of incubation, R76713 completely blocked the behavioral demasculinization of females without affecting the behavior of the males. After a treatment with T, almost all R76713-treated females showed as adults a masculine copulatory behavior that was undistinguishable from the behavior of intact males. This effect was fully reversed by the injection in egg of EB demonstrating that the effects of R76713 were specifically due to the suppression of endogenous estrogens. Injection of R76713 during the late phase of the incubation (Day 12 or Day 15) only maintained weak copulatory behavior in females which confirmed that the behavioral demasculinization in quail takes place mainly though not exclusively during the early stages of ontogeny. In a last experiment, we combined an early R76713 treatment with an injection of EB either on Day 9 or on Day 14 of incubation. This showed that the sensitivity to differentiating effects of estrogens varies with age in a sexually differentiated manner. The EB injection on Day 9 demasculinized both male and female embryos. If this injection was delayed until Day 14, it was no longer effective in males but still caused a partial demasculinization of females. This demonstrates that even if females are not yet behaviorally demasculinized on Day 9 of incubation (suppression of aromatase activity at that age will maintain the behavior), their sensitivity to estrogens is already different from that of males.