Endothelial-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 resistance uterine arteries of the rat and also analyzes the contribution of endothelial cell Ca²⁺ signaling to this process. A parallel study was also performed using mesenteric arteries to provide comparison with a non-reproductive vasculature. Mature female rats underwent ovariectomy, with one-half receiving 17ß-estradiol replacement (OVX+E) and the other half serving as estrogen-deficient controls (OVX). Uterine or mesenteric resistance arteries were harvested, cannulated, and pressurized. NO and prostacyclin production were inhibited with 200 µM LNNA and 10 µM indomethacin, respectively. ACh effectively dilated the arteries pre-constricted with phenylephrine, but failed to induce dilatation of vessels pre-constricted with high K⁺ solution. ACh EC₅₀ values were decreased by estrogen replacement by 5- and 2-fold in uterine and mesenteric arteries, respectively. As evidenced from fura 2-based measurements of endothelial cell cytoplasmic Ca²⁺ (EC [Ca²⁺]_i), estrogen replacement was associated with increased basal and ACh-stimulated EC [Ca²⁺]_i rise in uterine, but not in mesenteric vessels. These data demonstrate that EDHF contributes to endothelium-dependent vasodilation of resistance uterine and mesenteric arteries and that estrogen controls EDHF-related mechanism(s) more efficiently in reproductive vs. non-reproductive vessels. Enhanced endothelial Ca²⁺ signaling may be an important underlying mechanism in estrogenic modulation of EDHF-mediated vasodilation in small resistance uterine arteries.