Endothelial nitric oxide synthase (eNOS) plays an important role in control of vascular tone and angiogenesis among other functions. Its regulation is complex and has not been fully established. Several studies have emphasized the importance of phosphorylation in the regulation of eNOS activity. Although it is commonly accepted that protein kinase C (PKC) signaling inhibits eNOS activity by phosphorylating Thr⁴⁹⁷ and dephosphorylating Ser¹¹⁷⁹, the distinct role of different PKC isoforms has not been studied so far. The PKC family comprises roughly 12 different isozymes that activate distinct downstream pathways. The present study was designed to investigate the role of PKCα isoform in regulation of eNOS activity. Overexpression of PKCα in primary endothelial cells was associated with increased eNOS-Ser¹¹⁷⁹ phosphorylation and increased NO production. Inhibition of PKCα activity either by siRNA transfection or by overexpression of a dominant negative mutant resulted in a marked decrease in FGF2-induced Ser¹¹⁷⁹ phosphorylation and NO production. In vivo, PKCα transduction in rat femoral arteries resulted in a significant increase in the resting blood flow that was suppressed by treatment with l-NAME, an eNOS inhibitor. In conclusion, these data demonstrate for the first time that PKCα stimulates NO production in endothelial cells and plays a role in regulation of blood flow in vivo.