The endothelial NO synthase (eNOS) can be tyrosine phosphorylated, but the consequences of this modification on enzyme localisation and activity are not known. Tyrosine phosphorylation of eNOS in human endothelial cells could be enhanced by exposing cells to fluid shear stress (12 dynes cm-2) for 30 minutes. The shear stress-induced phosphorylation of eNOS correlated temporally with its association with the proline-rich tyrosine kinase 2 (PYK2). The eNOS immunoprecipitated from COS-7 cells overexpressing eNOS and PYK2 was tyrosine phosphorylated on Tyr657 (MALDI-TOF spectroscopy). NO production by HEK 293 cells over-expressing wild-type eNOS and PYK2 was approximately 50% of that in PYK2-deficient cells. Point mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) completely inactivated the enzyme, while the activity of a non-phosphorylatable mutant (phenylalanine) was comparable to that of the wild-type enzyme. The Tyr657E eNOS mutant, like the wild-type eNOS in cells exposed to shear stress was partly insoluble in Triton X-100 and localised with filamentous structures. These data indicate that PYK2 mediates eNOS tyrosine phosphorylation on Tyr657 following its activation by fluid shear stress and that this modification attenuates the activity of the enzyme.