Tyr83 in endothelial nitric oxide synthase (eNOS) has been identified previously as a site of Src kinase–mediated phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) that is phosphorylated in response to oxidant stress. In the present study, we have used a phospho-specific antibody to show that Tyr83 in eNOS is also phosphorylated in both BAECs and intact blood vessel segments in response to treatment with a variety of different eNOS-activating agonists, including thapsigargin, vascular endothelial growth factor, bradykinin, ATP, sphingosine-1-phosphate, estrogen, angiopoietin, and acetylcholine. Agonist stimulation of eNOS Tyr83 phosphorylation as well as agonist stimulation of endothelial NO release in BAECs is blocked by Src kinase inhibition by either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) or by dominant negative Src. Mutation of Tyr83 to a nonphosphorylatable Phe blocks agonist stimulation of NO release from eNOS-reconstituted eNOS knockdown endothelial cells. Mutation of Tyr83 also attenuates agonist-induced relaxation of eNOS-reconstituted aortic rings from eNOS knockout mice. Phosphorylation of eNOS at Tyr83 thus appears to be a common covalent modification that is induced, not only by oxidant stress but also by other physiologically relevant extracellular signals known to be important in regulation of eNOS activity in vivo. Moreover, our results demonstrate an important role for Src-mediated phosphorylation of eNOS at Tyr83 in agonist stimulation of eNOS activation and vascular relaxation.