It is now accepted that estrogens play a role in male fertility and that exposure to exogenous estrogens during fetal/neonatal life can lead to reproductive disorders in the male. However, the estrogen receptor (ER)-mediated processes involved in the regulation of male reproduction during fetal and neonatal development are still largely unclear. We previously reported that ERβ deficiency affects gametogenesis in mice but changes neither the number nor the differentiated functions of fetal Leydig cells. We show here that ERα-deficient mice (ERα^−/−) display higher levels of testicular testosterone secretion than wild-type mice from fetal d 13.5 onwards. This results from higher levels of steroidogenic activity per fetal Leydig cell, as indicated by the hypertrophy of these cells and the higher levels of mRNA for StAR, P450c17 and P450scc in the testis, for a similar number of Leydig cells. Because LH is not produced on fetal d 13.5 and because no change in plasma LH concentration was observed in 2-d-old ERα-deficient mice, LH is probably not involved in the effects of estrogens on testicular steroidogenesis in fetal and early neonatal Leydig cells. Furthermore, inactivation of ERβ did not change the effect of ERα inactivation on steroidogenesis. Lastly, in an organ culture system, 1 μm diethylstilbestrol decreased the testosterone secretion of wild-type fetal and neonatal testes but not of ERα^−/− testes. Thus, this study shows that endogenous estrogens physiologically inhibit steroidogenesis via ERα by acting directly on the testis early in fetal and neonatal development.