Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in the regulation of vascular microcirculatory tone. This study explores the role of estrogen in controlling EDHF-mediated vasodilation of uterine resistance arteries of the rat and also analyzes the contribution of endothelial cell (EC) Ca2+ signaling to this process. A parallel study was also performed with mesenteric arteries to provide comparison with a nonreproductive vasculature. Mature female rats underwent ovariectomy, with one half receiving 17β-estradiol replacement (OVX+E) and the other half serving as estrogen-deficient controls (OVX). Uterine or mesenteric resistance arteries were harvested, cannulated, and pressurized. Nitric oxide and prostacyclin production were inhibited with 200 μM NG-nitro-l-arginine and 10 μM indomethacin, respectively. ACh effectively dilated the arteries preconstricted with phenylephrine but failed to induce dilation of vessels preconstricted with high-K+ solution. ACh EC50 values were decreased by estrogen replacement by five- and twofold in uterine and mesenteric arteries, respectively. As evidenced by fura-2-based measurements of EC cytoplasmic Ca2+ concentration ([Ca2+]i), estrogen replacement was associated with increased basal and ACh-stimulated EC [Ca2+]i rise in uterine, but not mesenteric, vessels. These data demonstrate that EDHF contributes to endothelium-dependent vasodilation of uterine and mesenteric resistance arteries and that estrogen controls EDHF-related mechanism(s) more efficiently in reproductive vs. nonreproductive vessels. Enhanced endothelial Ca2+ signaling may be an important underlying mechanism in estrogenic modulation of EDHF-mediated vasodilation in small resistance uterine arteries.