Aim: The redox-sensitive proline-rich tyrosine kinase Pyk2 phosphorylates eNOS on tyrosine 657, thus abrogating eNOS activity. Since Pyk2 is also known to be activated by angiotensin (Ang) II and reactive oxygen species, we investigated whether this mechanism plays a role in the loss of NO bioavailability in pathologies associated with increased Ang II and H2O2 levels. Methods & Results: Western blotting of Tyr phosphorylated proteins from native murine lung endothelial cells showed that AngII (100 nM) activated Pyk2 and enhanced the phosphorylation of eNOS on Tyr657. The Tyr657 phosphorylation of eNOS depended on Nox2, Pyk2, and H2O2 since it was prevented in Nox2-deficient cells, by Pyk2 siRNA and by catalase, respectively. A low dose of H2O2 (30 mM) mimicked the effect of AngII on eNOS Tyr657 phosphorylation in cultured cells as well as in isolated arteries. At this concentration, H2O2 limited basal as well as agonist-induced NO production in cells (measured by cGMP RIA) and impaired acetylcholine-induced vasodilatation in murine aortae (72±10 vs. 87±6% relaxation, P=0.009). Adenoviral overexpression of a dominant negative Pyk2 mutant in carotid arteries protected 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, but vessels expressing the non-phosphorylatable eNOS- Tyr657Phe were resistant to H2O2. Finally, chronic (3 week) treatment of wild-type, but not Nox2-/- mice with AngII (1 mg/kg/day) also caused a decrease in endothelium-dependent relaxation accompanied by increased phosphorylation of eNOS on Tyr657. Conclusion: Pyk2 activation by AngII 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 many cardiovascular diseases.