Estrogen receptors are direct regulators of transcription that function by binding to specific DNA sequences in promoters of target genes. The two cloned forms of estrogen receptors, alpha and beta, are expressed in the central nervous system by different neuronal populations. Astrocytes in vitro are also reported to express estrogen receptor alpha; however, this expression has not been confirmed in the rat brain in vivo. The apparent absence of estrogen receptors in glia in vivo contrasts with the well-known effects of this hormone on astrocytes of different brain areas, including the hippocampal formation. In this study, the expression of estrogen receptors in the hippocampal formation of adult male rats has been assessed by confocal microscopy. Estrogen receptor alpha-immunoreactivity was localized in neuronal nuclei in the pyramidal cell layer of CA1-CA3 fields. Estrogen receptor beta-immunoreactivity was observed in the perikarya, apical dendrites, and cell nuclei of pyramidal neurons in CA1 and CA2. Furthermore, estrogen receptor beta-immunoreactive glia were observed in CA1, CA2, CA3, and in the hilus of the dentate gyrus of male and female rats. Estrogen receptor beta-immunoreactivity was localized in glial processes and perikarya and, in some cases, in glial cell nuclei. Double immunocytochemical labeling of estrogen receptor beta and the specific astroglial marker glial fibrillary acidic protein revealed that estrogen receptor beta-immunoreactive glial cells were astrocytes. Estrogen receptor alpha was not co-localized with glial fibrillary acidic protein. The presence of estrogen receptor beta in astrocytes of adult male and female rats demonstrates a possible mechanism by which estrogen can directly modulate gene expression in these cells.