Although the distribution of estrogen receptor β (ERβ) immunoreactivity in the rat central nervous has been reported, no such data are available in the mouse. The present study used in vivo autoradiography utilizing a 125I-estrogen that has equal binding affinity for both receptors as well as immunohistochemistry for ERβ and ERα, to investigate and compare the distribution of the two ERs in the mouse CNS. The use specific antisera against ERα and ERβ allowed us to evaluate the contribution of these receptors to the binding detected with autoradiography. In addition, data were collected in ovariectomized wildtype and ERα KO (knockout) mice to examine developmental regulation of ERβ expression by ERα. These studies revealed that in the mouse CNS, combining immunoreactivity for ERα with that for ERβ accounted for all regions where binding was seen using autoradiography. Therefore, these data strongly suggest that the major contributors of estrogen binding in the mouse CNS are ERα and ERβ. Together, these data provide an anatomical foundation for future studies and advance our understanding of estrogen action in the CNS. Moreover, since the immunocytochemical images were similar in wildtype and ERα KO mice, these studies suggest that the lack of ERα does not influence the expression of ERβ in the central nervous system. J. Comp. Neurol. 473:270–291, 2004. © 2004 Wiley-Liss, Inc.