We have examined mechanisms for PGE2-mediated vasodilatation and hypothesized an involvement of endothelial NO synthase (eNOS). Aortic rings from mice were used for contraction studies. Blood pressure changes in response to PGE2 were measured in conscious mice. Single doses of PGE2 caused concentration-dependent relaxations during contractions to phenylephrine (EC50 =5 × 10^-8 mol/L). Relaxation after PGE2 was absent in rings without endothelium, in rings from eNOS-/- mice and was abolished by L-NAME and by inhibition of guanylate cyclase. Vascular cGMP, but not cAMP, content increased after PGE2. PGE2- 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. Relaxation was similar to control in rings from EP2-/-. Inhibitors of the cAMP-PKA pathway attenuated, while the inhibitor of protein phosphatase 1C, calyculin (10^-8 mol/L), abolished the PGE2-mediated relaxation. In aortic rings, PGE2 dephosphorylated eNOS at Thr.495 Chronically catheterized eNOS-/- mice were hypertensive (137 mmHg3.6, n = 13 vs. 101 mmHg3.9, n = 9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE2 compared to wild type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE2 elicits EP4 receptor-mediated, endothelium- dependent stimulation of eNOS activity by dephosphorylation at Thr495 resulting in guanylyl cyclase-dependent vasorelaxation and accumulation of cGMP in aortic rings. We speculate that activation of eNOS and NO release may be a common feature of agonists that elicit cAMP formation in the endothelium.