Sexual differentiation in the amniote brain is believed to be regulated by gonadal sex steroid hormones. Recently, however, the possibility of brain-autonomous sexual differentiation in avian and reptilian species has been reported. We conducted here an expressional analysis of genes related to sex steroid hormones in the chick-embryo brain before gonadal sexual differentiation. Female-specific P450 aromatase expression in the gonad was observed at day 6.5 of incubation, as previously reported, whereas the mRNAs of cholesterol side-chain cleavage enzyme, androgen receptor, and estrogen receptors α and β were clearly expressed in all brain samples of both male and female embryos from day 4.5 of incubation. P450 aromatase was expressed in some brain samples before day 5.5 of incubation and in all brain samples after day 6 of incubation. The mRNA of Ad4BP/SF-1, a transcription factor that regulates steroidogenic enzymes, showed higher expression levels in the male brain than in the female brain at day 5.5 of incubation. This gene was expressed in the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Embryonic Ad4BP/SF-1 expression is reported to play an important role in the formation of this region. These results therefore suggest the involvement of a sex steroid hormone signaling system in brain-autonomous sexual differentiation.