During the follicular phase of the ovarian cycle, when the local estrogen-to-progesterone ratio is elevated, uterine blood flow is elevated. This vasodilatory response is reproduced by exogenous 17β-estradiol (E2β) administration via a nitric oxide (NO)-mediated mechanism. We hypothesized that endogenous ovarian estrogen and exogenous E2β treatment elevate expression of endothelial cell-derived NO synthase (eNOS) in uterine, but not in systemic, arteries. Uterine, mammary, and systemic (renal and/or omental) arteries were collected from 1) ewes synchronized to the follicular ( day −1 to day 0) or luteal ( day 10) phases of the ovarian cycle ( n = 4 per phase), 2) ovariectomized ewes 120 min after systemic vehicle or E2β (5 μg/kg iv) treatment, and 3) ovariectomized ewes on days 0, 3, 6, 8, and 10 of E2β (5 μg/kg iv, followed by 6 μg/kg per day) treatment. Expression of eNOS was localized primarily to the endothelium rather than vascular smooth muscle (VSM) in all arteries examined by immunohistochemistry and Western analysis; inducible NOS was not detected in either endothelium or VSM. Expression of eNOS protein was greater ( P < 0.05) in uterine, but not in systemic, artery endothelium-isolated protein collected from follicular versus luteal phase ewes. Acute systemic E2β treatment of ovariectomized ewes increased ( P < 0.05) eNOS protein levels in uterine artery endothelium. Prolonged E2β administration progressively increased uterine, but not systemic, artery endothelial eNOS protein expression. Therefore, the increased local estrogen-to-progesterone ratio during the follicular phase locally elevates eNOS expression, which possibly elevates uterine blood flow. These responses can be partly reproduced with E2β administration.