The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca(2+) by the phosphorylation of Ser(1177) but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca(2+)-dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr(495) in the calmodulin (CaM)-binding domain. eNOS Thr(495) was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation. In the same cells, bradykinin enhanced the phosphorylation of Ser(1177), which was maximal after 5 minutes, and abolished by the CaM-dependent kinase II (CaMKII) inhibitor KN-93. Bradykinin also enhanced the association of CaMKII with eNOS. Phosphorylation of Thr(495) was attenuated by the protein kinase C (PKC) inhibitor Ro 31-8220 and after PKC downregulation using phorbol 12-myristate 13-acetate. The agonist-induced dephosphorylation of Thr(495) was completely Ca(2+)-dependent and inhibited by the PP1 inhibitor calyculin A. Little CaM was bound to eNOS immunoprecipitated from unstimulated cells, but the agonist-induced dephosphorylation of Thr(495) enhanced the association of CaM. Mutation of Thr(495) to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp(495) mutant bound almost no CaM. Accordingly, NO production by the Ala(495) mutant was more sensitive to Ca(2+)/CaM than the aspartate mutant. These results suggest that the dual phosphorylation of Ser(1177) and Thr(495) determines the activity of eNOS in agonist-stimulated endothelial cells. Moreover, the dephosphorylation of Thr(495) by PP1 precedes the phosphorylation of Ser(1177) by CaMKII. The full text of this article is available at http://www.circresaha.org.