In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH4 is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and generation are unknown. Therefore, we phosphorylated recombinant BH4-free eNOS in vitro using native kinases and determined generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca2+ sensitivity of eNOS, such that generation became largely Ca2+-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase C (PKC) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of and NO generation, altering both the Ca2+ sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases generation from the enzyme at low Ca2+ concentrations, and PKC-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.