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Our previous work showed that chronic activation of the membrane-bound estrogen receptor GPR30/GPER significantly lowers blood pressure in ovariectomized hypertensive mRen2.Lewis female rats which may, in part, reflect direct vasodilatory actions. The current study assessed the hypothesis that cyclic adenosine monophosphate (cAMP) signaling contributes to GPER-mediated vasorelaxation. In mesenteric resistance arteries from intact Lewis females, relaxation to 17-β-estradiol (E2; 47 ± 3% of phenylephrine contraction vs. vehicle 89 ± 2%, P < 0.001) or G-1 (44 ± 8%, P < 0.001) was blunted to a similar extent by denuding (P < 0.001) or the nitric oxide synthase inhibitor l-NAME (P < 0.001). In contrast, the cyclooxygenase inhibitor indomethacin did not alter vasodilation (P > 0.05). The cAMP analog Rp-cAMPS partially attenuated vasodilation (65 ± 7%, P < 0.001), while the combination of l-NAME and Rp …