The present experiments were designed to test the hypothesis that prostaglandin (PG) E(2) causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (EP) receptors were used for contraction studies. Blood pressure changes in response to PGE(2) were measured in conscious mice. Single doses of PGE(2) caused concentration-dependent relaxations during contractions to phenylephrine (EC(50)=5*10(-8) mol/L). Relaxation after PGE(2) was absent in rings without endothelium and in rings from eNOS(-/-) mice and was abolished by N(G)-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H(1,2,4)-oxadiazolo-[4,3-a]quinoxalin-1-one. In PGE(2)-relaxed aortic rings, the cGMP content increased significantly. PGE(2)-induced relaxations were abolished by the EP4 receptor antagonist AE3-208 (10(-8) mol/L) and mimicked by an EP4 agonist (AE1-329, 10(-7) mol/L) in the presence of endothelium and eNOS only. Relaxations were attenuated significantly in rings from EP4(-/-) mice but normal in EP2(-/-). Inhibitors of the cAMP-protein kinase A pathway attenuated, whereas the inhibitor of protein phosphatase 1C, calyculin (10(-8) mol/L), abolished the PGE(2)-mediated relaxation. In aortic rings, PGE(2) dephosphorylated eNOS at Thr(495). Chronically catheterized eNOS(-/-) mice were hypertensive (137+/-3.6 mm Hg, n=13, versus 101+/-3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE(2) compared with wild-type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE(2) elicits EP4 receptor-mediated, endothelium-dependent stimulation of eNOS activity by dephosphorylation at Thr(495) resulting in guanylyl cyclase-dependent vasorelaxation and accumulation of cGMP in aortic rings.