Pregnancy and the follicular phase are physiological states of elevated oestrogen 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. Oestrogen exerts its vasodilatory effect by binding to its specific oestrogen 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 versus VSM ERalpha and ERbeta expression in uterine arteries (UAs) during the ovarian cycle, pregnancy and with exogenous hormone replacement therapy (HRT) are currently unknown. ER mRNA and protein localization was determined by in situ hybridization (ISH) using 35S-labelled riboprobes and immunohistochemistry (IHC), respectively. UA endothelial (UAendo), UA VSM, omental artery endothelium (OA endo), and OA VSM proteins were isolated and ERalpha and ERbeta protein expression was determined by Western analysis. We observed by ISH and IHC that ERalpha and ERbeta mRNA and protein were localized in both UAendo and UA VSM. Immunoblot data demonstrated ovarian hormone specific regulation of ERalpha and ERbeta protein in UAendo and UA VSM. Compared to luteal phase sheep, both ERalpha and ERbeta levels in UAendo were elevated in follicular phase sheep. Whereas ERbeta was elevated by pregnancy in UAendo and UA VSM, ERalpha was not appreciably altered. eNOS was increased in UAendo from follicular and pregnant sheep. Ovariectomized ewes (OVEX) had substantially reduced UAendo ERbeta, but not UAendo ERalpha or OAendo ERalpha and ERbeta. In contrast, OVEX increased UA VSM ERalpha and ERbeta and decreased OA VSM ERalpha and ERbeta. Treatment with oestradiol-17beta (E2beta), but not progesterone or their combination, increased UAendo ERalpha levels. The reduced ERbeta in UAendo from OVEX ewes was reversed by E(2)beta and progesterone treatment. While ERalpha and eNOS were not elevated in any other reproductive or non-reproductive endothelia tested, ERbeta was augmented by pregnancy in uterine, mammary, placenta, and coronary artery endothelia. ERalpha and ERbeta 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 oestrogen 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.