The Angiotensin II type 2 receptor (AT 2R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT 2R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT 2R-mediated phosphorylation of eNOS at Ser 1177, activation of phosphatases and dephosphorylation of eNOS at Tyr 657 and Thr 495 are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT 2R-agonist Compound 21 (C21) (1 μM) in the presence or absence of either PD123319 (10 μM; AT 2R antagonist), L-NG-Nitroarginine methyl ester (L-NAME) (10 μM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na 3VO 4) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser 1177) or inhibitory eNOS residues (eNOS-Tyr 657, eNOS-Thr 495) was determined by Western blotting. Phosphorylation of Akt at Ser 473 was measured to estimate Akt activity. AT 2R stimulation significantly increased NO release from HAEC, which was blocked by PD123319, L-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT 2R stimulation resulted in phosphorylation of eNOS-Ser 1177 and dephosphorylation of eNOS-Tyr 657 and eNOS-Thr 495. Phosphorylation at eNOS-Ser 1177 was prevented by inhibition of Akt with MK-2206. From these data, we conclude that AT 2R stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser 1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr 657, eNOS-Thr 495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.