Studies on the posttranslational regulation of the endothelial NO synthase (eNOS) have long focused mainly on its activation through phosphorylation of serine and threonine residues. Particularly the phosphorylation of Ser1177 is generally acknowledged as being critical for eNOS activity. We observed, however, that one of the physiologically most relevant stimuli for eNOS activation, i.e. fluid shear stress, elicits phosphorylation not only of Ser1177, but also of tyrosine residues. Further studies revealed that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2), a kinase abundantly expressed in native and primary cultures of human endothelial cells that phosphorylates eNOS on Tyr657. However, the PYK2-dependent phosphorylation of eNOS-Y657 abolished enzyme activity completely, preventing both NO and superoxide production. Indeed, in arteries overexpressing a constitutively active PYK2 mutant flow- and acetylcholine-induced vasodilatation were severely impeded. PYK2 is a redox sensitive kinase that can also be activated by angiotensin (Ang) II and reactive oxygen species and we found that Ang II enhances the tyrosine phosphorylation of eNOS in an AT₁, H₂O₂ and PYK2-dependent manner. Low concentrations (1–100 mmol/L) of H₂O₂ stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated murine aortae, H₂O₂ (30 mmol/L) induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant negative PYK2 mutant protected against H₂O₂-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS^-/- mice overexpressing the non-phosphorylatable eNOS Y657F mutant were also protected against H₂O₂. In vivo three weeks infusion of Ang II, a treatment known to significantly elevate vascular redox stress, considerably increased levels of Tyr657 phosphorylated eNOS in aortae and this was associated with a clear impairment in endothelium-dependent vasodilatation. Taken together, endothelial PYK2 activation in situations of physiological (fluid shear stress) or pathophysiological (oxidative) stress causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. Activation of PYK2 by fluid shear stress attenuates the activity of eNOS despite persistent Ser1177 phosphorylation. We postulate that this mechanism explains the low, but sustained NO production observed in fluid shear stress and thus serves as a protective mechanism preventing eNOS uncoupling due to cofactor depletion. However, in situations of elevated redox stress, PYK2-dependent eNOS-Y657 phosphorylation may also impede NO production and thus contribute to endothelial dysfunction.