—The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca²⁺ by the phosphorylation of Ser¹¹⁷⁷ but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca²⁺-dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr⁴⁹⁵ in the calmodulin (CaM)-binding domain. eNOS Thr⁴⁹⁵ was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation. In the same cells, bradykinin enhanced the phosphorylation of Ser¹¹⁷⁷, 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 Thr⁴⁹⁵ 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 Thr⁴⁹⁵ was completely Ca²⁺-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 Thr⁴⁹⁵ enhanced the association of CaM. Mutation of Thr⁴⁹⁵ to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp⁴⁹⁵ mutant bound almost no CaM. Accordingly, NO production by the Ala⁴⁹⁵ mutant was more sensitive to Ca²⁺/CaM than the aspartate mutant. These results suggest that the dual phosphorylation of Ser¹¹⁷⁷ and Thr⁴⁹⁵ determines the activity of eNOS in agonist-stimulated endothelial cells. Moreover, the dephosphorylation of Thr⁴⁹⁵ by PP1 precedes the phosphorylation of Ser¹¹⁷⁷ by CaMKII. The full text of this article is available at http://www.circresaha.org.