Estrogen interacts with estrogen receptors (ERs) to induce vasodilation, but the ER subtype and post-ER relaxation pathways are unclear. We tested if ER subtypes mediate distinct vasodilator and intracellular free Ca 21 concentration ([Ca 21 ] i ) responses via specific relaxation pathways in the endothelium and vascular smooth muscle (VSM). Pressurized mesenteric microvessels from female Sprague-Dawley rats were loaded with fura-2, and the changes in diameter and [Ca 21 ] i in response to 17b-estradiol (E2) (all ERs), PPT (4,49,-pyrazole-1,3,5-triyl]-tris-phenol) (ERa), diarylpropionitrile (DPN) (ERb), and G1 [(6)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro:3H-cyclopenta(c)quinolin-8-yl]-ethanon] (GPR30) were measured. In microvessels preconstricted with phenylephrine, ER agonists caused relaxation and decrease in [Ca 21 ] i that were with E2 5 PPT . DPN . G1, suggesting that E2-induced vasodilation involves ERa . ERb . GPR30. Acetylcholine caused vasodilation and decreased [Ca 21 ] i , which were abolished by endothelium removal or treatment with the nitric oxide synthase blocker N v -nitro-L-arginine methyl ester (L-NAME) and the K 1 channel blockers tetraethylammonium (nonspecific)