Determination of the specific roles of the estrogen receptor (ER) forms in reproductive processes of different species remains incomplete. In the present experiment, cellular localization and changes in relative amounts of the ERα and ERβ in late developing ovarian follicles, oviduct, and uterus were determined during the follicular phase of the estrous cycle in sheep. Ewes in mid-luteal phase were treated with prostaglandin F(2α) (PG) to induce luteolysis and control the onset of the follicular phase. The oviducts, uterus, and the ovaries were collected at 0 (ewes not treated with PG), 4, 18, and 36 h after PG treatment (early, mid, and late follicular phase, respectively) and processed to evaluate the ERs using immunohistochemical (IHC) procedures. The ERα was localized to nuclei of granulosa cells of late developing follicles and most cells of the oviduct and uterus. The ERβ was detected only in ovarian follicles using two antibodies directed to different regions of the ERβ. Western immunoblotting demonstrated that the antibody directed against the N-terminal region of the ERβ detected one isoform (approximately 53 kDa) whereas the antibody directed against the C-terminus detected two ERβ isoforms (approximately 53 kDa and 59 kDa). Western and IHC results combined indicated presence of the 59 kDa ERβ in granulosa cells and the 53 kDa ERβ in both granulosa and theca cells. Relative amounts (immunostaining intensity) of the ERα increased (P<.05) in granulosa cells of preovulatory follicles and in the isthmian muscularis of the oviduct at the late follicular phase. Amounts of the ERα in the mucosal epithelium of the oviductal regions (isthmus, ampulla, and infundibulum), and in various uterine cell types (glandular and luminal epithelia, endometrial stromal cells, and myometrium) did not change (P>.05) throughout the follicular phase. A major increase (four-fold) in expression of the 53 kDa ERβ in the theca and a less pronounced increase in the granulosa occurred at the late follicular phase. The ERα is broadly expressed in reproductive organs of sheep and is upregulated only in few cell types during the late follicular phase. Immunoreactive ERβ was detected only in the ovary. Important estrogen actions in theca cells during preovulatory follicular development likely occur in association with a major increase in expression of an ERβ isoform.