Objective: Metabolic syndrome entails hypertension, hyperglycemia, obesity and hypercholesterolemia. This syndrome increases the risk of cardiovascular disease and diabetes. Hyperglycemia during coronary reperfusion is associated with a poor prognosis. Contrastingly, targeting correction of hyperglycemia in clinical trials has not improved clinical outcome or has even been detrimental. H2 O2 is produced under hyperglycemic conditions and under reperfusion. This study aims to provide a mechanistic approach evaluating the impact of high glucose on the endothelial nitric oxide pathway in a H2 O2 -rich environment. Methods and results: HUVECs (human umbilical vein endothelial cells) were exposed to high glucose (20 mM) for either 20 or 72 hours co-incubated with or without H2 O2 (400 µM) for 30 minutes as models of increased oxidative stress during acute and prolonged hyperglycemia, respectively. The presence of reactive oxygen species (ROS) in both mitochondria and cytoplasm was measured by fluorescence activated cell sorting (FACS). Phosphorylation of endothelial nitric oxide synthase (eNOS) on threonine 495 (Thr495) and serine 1177 (Ser1177) was assessed by western blotting. Short-term (20 hours) high concentration of glucose alone increased ROS in mitochondria to 133.5% (p<0.05), whereas prolonged (72 hours) did not increase mitochondrial ROS. The increase in mitochondrial ROS could be attenuated by the anti-oxidant N-acetyl-L-cysteine (NAC). Incubation with H2 O2 for 30 minutes resulted in an increase in Thr495 phosphorylation (to 425%, p<0.01) and a decrease in Ser1177 phosphorylation (to 50.6%, p<0.01). Preincubation for 20 hours with 10 and 20 mM glucose did not affect phosphorylation of Thr495 and Ser1177. Stimulating HUVECs that were pre-incubated with 20 mM glucose for 72 hours with H2 O2 increased Thr495 phosphorylation to 146.6% (p<0.05). PKC inhibition attenuated the H2 O2 -induced Thr495 phosphorylation in cells incubated with high glucose levels for 72 hours. Conclusion: Acute exposure to high glucose induces oxidative stress. H2 O2 leads to phosphorylation of eNOS at Thr495 and dephosphorylation of Ser1177. After prolonged exposure to high glucose levels, the addition of H2 O2 yields phosphorylation of Thr495 through the PKC pathway