Pregnancy and the follicular phase are physiologic states of elevated estrogen levels and rises in uterine blood flow (UBF). The dramatic increase in utero- placental blood flow during gestation is required for normal fetal growth and development. Estrogen exerts its vasodilatory effect by binding to its specific estrogen receptors (ER) in target cells, resulting in increased expression and activity of endothelial nitric oxide synthase (eNOS) to relax vascular smooth muscle (VSM). However, the regulation of endothelial vs. VSM ER& [alpha] and ER expression in uterine arteries (UAs) during the ovarian cycle, pregnancy and with exogenous hormone replacement therapy (HRT) are currently unknown. Methods: ER mRNA and protein localization was determined by in situ hybridization (ISH) using [35S]-labeled riboprobes and immunohistochemistry (IHC), respectively. UA endothelial (UAendo), UA VSM, omental artery (OA endo), and OA VSM proteins were isolated and ERa and ERb protein expression was determined by Western analysis. Results: We observed by ISH and IHC that ER and ER mRNA and protein were localized in both UAendo and UA VSM. Immunoblot data demonstrated ovarian hormone specific regulation of ER and ER protein in UAendo and UA VSM. Compared to luteal phase sheep, both ER and ER levels in UAendo were elevated in follicular phase sheep. Whereas ER was elevated by pregnancy in UAendo and UA VSM; ER& [alpha] was not appreciably altered. eNOS was increased in UAendo from follicular and pregnant sheep. Ovariectomized ewes (OVEX) had substantially reduced UAendo ER, but not UAendo ER or OAendo ER& [alpha] and ER. In contrast, OVEX increased UA VSM ER and ER and decreased OA VSM ER& [alpha] and ER. Treatment with estradiol-17& [beta] (E2), but not progesterone or their combination increased UAendo ER levels. The reduced ER in UAendo from OVEX ewes was reversed by E2 and progesterone treatment. While ER& [alpha] and eNOS were not elevated in any other reproductive or non-reproductive endothelia tested, ER& [beta] was augmented by pregnancy in uterine, mammary, placenta, and coronary artery endothelia. Conclusion: ER and ER mRNA and protein are expressed in UA endothelium with expression levels depending on the endocrine status of the animal, indicating UA endothelium is a target for estrogen action in vivo, and that the two receptors appear to be differentially regulated in a spatial and temporal fashion with regard to the reproductive status or HRT.