To study the effects of gestational exposure to estrogen on early gonadal differentiation, pregnant mice were treated by sc injection of diethylstilbestrol (DES) or vehicle from embryonic day (E) 8.5 to E14.5, and gonads at E11.5, E12.5, and E14.5 were examined. Quantitative real-time RT-PCR and in situ hybridization revealed that mRNA levels of steroidogenic factor 1 (SF-1), a key regulator of gonadal differentiation, and several male gonad-specific genes, including Müllerian-inhibiting substance (MIS), the steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage cytochrome P450 (P450scc) and Cerebellin 1 precursor protein (Cbln 1), were significantly decreased in the DES-treated testis compared with the control testis at E12.5 and/or E14.5. Immunohistochemistry demonstrated that the staining intensities for SF-1 and MIS in Sertoli cells were apparently reduced in the DES-treated testis compared with those of the controls at E12.5 and E14.5. Because MIS, StAR, P450scc, and Cbln 1 are activated under the regulation of SF-1, the down-regulation of these factors may be due to reduced SF-1 expression. Immunohistochemistry for laminin-1 demonstrated that ovigerous cords in the DES-treated ovary were smaller than those in controls at E14.5. Moreover, the number of 5-bromo-2'deoxyuridine-5-monophosphate-labeled cells in the DES-treated testis was significantly reduced at E12.5 and E14.5 compared with controls, and that in the DES-treated ovary remained higher than that in the control ovary at E14.5. The results suggest that exogenous estrogens can alter sex-specific genetic pathways governing early differentiation and cell proliferation of both male and female gonads.