In the present study we tested the hypothesis that PGE2 causes vasodilatation by activation of eNOS using mouse aortic rings. Experiments were performed in isolated segments of mouse aorta suspended in a Mulvany-Halpern myograph. Phenylephrine (PE, 10^­5 M) followed by Acetylcholine (ACh, 10^­6 M) was added at the start of each experiment to test viability of the smooth muscle and the endothelium, respectively. PGE2, 10^­7 M caused 50.7 ± 6.4 % vasorelaxation of PE-constricted aortic rings. The PGE2- mediated relaxation was blocked by the NO- synthase inhibitor L-NAME, 10^­4 M and by the inhibitor of soluble guanylate cyclase, ODQ, 10^­6 M. The PGE2-mediated relaxation was absent in segments without endothelium and in the aorta from eNOS^-/- mice. The EP4-receptor blocker AE3- 208, 10^­8 M abolished the PGE2-mediated relaxation while the EP4 agonist AE1-329, 10^­7 M mimicked the effect of PGE2. Butaprost, an EP2- agonist had no effect on vasoreactivity and PGE2 dilated rings from EP2^-/- mice. The PGE2- mediated relaxation was significantly attenuated in rings from EP4^-/- mice. Three different blockers of the PKA pathway: Rp-8-Br-cAMPs, 10^­4 M, SQ22536, 10^­4 M and KT5720, 10^­6 M significantly diminished while the PP1-inhibitor Calyculin A, 10^­8 M completely abolished the PGE2-mediated vasorelaxation. In response to PGE2, the cGMP content of the rings increased significantly. Western Blot studies showed that exposure of preconstricted aortic rings to PGE2 caused significant dephosphorylation at Thr495 while Ser1177 phospho-eNOS did not change significantly. In summary, PGE2 elicits EP4 receptor-mediated, endothelium-dependent, stimulation of eNOS activity by dephosphorylation at Thr495 resulting in cGMP accumulation in vascular smooth muscle.