Spatiotemporal expression patterns of ER-alpha and ER-beta protein and mRNA in hamster ovarian cells during the estrous cycle and following hypophysectomy and selective hormone replacement were evaluated by immunofluorescence, immunoblotting and in situ hybridization analyses. Whereas ER-beta mRNA and protein expression predominated in granulosa cells and ER-alpha expression was in interstitial and thecal cells, overlap in receptor subtype expression across cell types was evident. Both ER subtypes were present from primordial follicle stage onward. ER-alpha mRNA levels and immunoreactivity started increasing from D3:0900 h in interstitial and granulosa cells and peaked on the proestrous (D4:0900 h). Regionalized higher expression of ER-alpha in granulosa cells in and around the forming antrum was evident. Surface epithelial cells were also positive. ER-beta mRNA and protein expression increased markedly in granulosa and interstitial cells on D2:0900 h, reached a peak on D3:0900 h, and then declined sharply on D4:0900 h. No change in ER expression occurred following the preovulatory gonadotropin surge. Whereas FSH or human CG stimulated ER-alpha mRNA and protein expression in hypophysectomized hamsters, only FSH could stimulate ER-beta mRNA and protein, and the effect was significantly attenuated by human CG. ER expression was stimulated by estrogen, but progesterone strongly inhibited estrogen action. These results indicate that ER expression is cell type specific to the larger extent and is critically regulated by reproductive hormones.