In endothelial cells, the proline-rich tyrosine kinase PYK2 is activated by fluid shear stress and phosphorylates eNOS on tyrosine 657. Tyrosine phosphorylation of eNOS inhibits enzyme activity and limits NO production in response to shear stress. Overexpression of PYK2 in isolated arteries abrogated NO-mediated vasodilatation. In atherosclerotic lesions, eNOS is partially inactive even in the presence of sufficient co-factors. Since PYK2 is also known to be activated by angiotensin (Ang) II and reactive oxygen species, we speculated that this mechanism plays a role in the loss of NO bioavailability in pathologies associated with increased Ang II and H₂O₂ levels. In native porcine aortic endothelial cells and in freshly isolated murine aortae, Ang II and H₂O₂ both enhanced the phosphorylation of eNOS on Tyr657. This effect was correlated with increased PYK2 activation. In cultured endothelial cells, low concentrations of H₂O₂ (1 - 100 mmol/L) stimulated the phosphorylation of Tyr657 without affecting that of either Ser1177 or Thr495. At these concentrations, H₂O₂ also limited basal NO production and markedly impaired NO production in response to bradykinin and ionomycin. Furthermore, low (30 mmol/L) concentrations of H₂O₂ impaired acetylcholine-induced vasodilatation in murine aorta. Adenoviral overexpression of a dominant negative PYK2 mutant in carotid arteries protected these arteries against oxidative impairment of endothelial function. Likewise, H₂O₂ diminished the endothelium-dependent relaxation in carotid arteries from eNOS^-/- mice infected with an adenovirus expressing wild-type eNOS, whereas vessels expressing the non-phosphorylatable eNOS-Tyr657Phe were resistant to the effect of H₂O₂. Finally, chronic (3 week) treatment of mice with Ang II (1 mg/kg/day) also caused a decrease in endothelium-dependent relaxation accompanied by increased phosphorylation of eNOS on Tyr657. Taken together, PYK2 activation under pathological conditions, i.e. increased Ang II and H₂O₂ causes the phosphorylation of eNOS on Tyr657 impeding NO production and endothelium dependent vasodilatation. This mechanism, in addition to eNOS uncoupling, may underlie the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin-angiotensin system and elevated redox stress.