Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser, and a negative regulatory site, Thr, in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr. It had a minimal effect on Ser phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser with no noticeable effect on Thr. Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A Gq inhibitor blocked thrombin- and SLIGRL-induced Ser phosphorylation, whereas it enhanced thrombin-induced Thr phosphorylation. In contrast, a G12/13 inhibitor blocked thrombin- and TFLLR-induced Thr phosphorylation, whereas it enhanced the Ser phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G12/13 and Gq, respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.