Recent studies demonstrating a close relationship between postprandial hyperglycemia and the incidence of atherosclerotic cardiovascular disease prompted us to investigate the generation and source of reactive oxygen species (ROS) in endothelial cells stimulated by short-term exposure to a high glucose concentration. In addition, we investigated the effect of insulin on ROS production induced by high glucose concentration. Cultured bovine aortic endothelial cells demonstrated a significant increase in intracellular ROS generation after a 3-h exposure to 25 mM glucose (131.4% versus 5 mM glucose). This increased generation of ROS was suppressed by an inhibitor of NAD(P)H oxidase. Intracellular ROS production in cells exposed to 3 h of high glucose concentration was increased significantly by the presence of a physiological concentration of insulin. However, after a 1-h exposure to high glucose levels, ROS generation in cells incubated with insulin was only about 80% of that measured in cells incubated without insulin. The generation of intracellular nitric oxide (NO) resulting from an acute insulin effect may account for this difference. In conclusion, acute hyperglycemia itself may possibly cause endothelial oxidative stress in patients with postprandial hyperglycemia. Endothelial oxidative stress may be determined by the interaction between NO and superoxide generation.