Rapid uterine vasodilatation following estrogen administration is believed to be mediated by endothelial production of nitric oxide (NO) via endothelial NO synthase (eNOS). However, the mechanism(s) by which estrogen activates eNOS in uterine artery endothelial cells (UAEC) is unknown. In this study, we observed that E2 and E2-BSA rapidly (<2 min) increased total NO_x production in UAEC in vitro. This was associated with rapid eNOS phosphorylation and activation, but was unaltered by pretreatment with actinomycin-D. ER protein was detectable in isolated plasma membrane proteins by immunoblotting and E2-BSA-FITC binding was evident on the plasma membrane of UAEC. E2 did not mobilize intracellular Ca2+, but E2 and ionomycin in combination induced greater eNOS phosphorylation than either E2 or ionomycin alone. E2 did not stimulate rapid Akt phosphorylation. E2 stimulated rapid ERK2/1 activation in a time- and dose-dependent manner with maximal responses observed at 5-10 min with E2 (10 nM to 1 µM) treatment. Acute activation of eNOS and NO_x production by E2 could be inhibited by PD98059 but not by LY294002. When E2-BSA was applied, similar responses in NO_x production, eNOS and ERK2/1 activation to these of E2 were achieved. In addition, E2 and E2-BSA induced ERK2/1 activation and ICI 182,780 could inhibit NOx production. Thus, acute activation of eNOS to produce NO in UAEC by estrogen is at least partially through an ERK pathway possibly via ER localized on the plasma membrane. This pathway may provide a novel mechanism for NO-mediated rapid uterine vasodilatation by estrogen.