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 PKCalpha isoform in regulation of eNOS activity. Overexpression of PKCalpha in primary endothelial cells was associated with increased eNOS-Ser1179 phosphorylation and increased NO production. Inhibition of PKCalpha 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, PKCalpha 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 PKCalpha stimulates NO production in endothelial cells and plays a role in regulation of blood flow in vivo.