It has been reported that oxidative stress is increased in vivo in the diabetic state. Increased oxidative stress is caused not only by accelerated production of oxygen-free radicals but also by decreased scavenging of those molecules. Endothelial cells are extremely sensitive to oxidative stress, resulting in impairments of various endothelial cell function. In this report, we studied the association of intracellular glucose metabolism and oxygen radical scavenging function via the glutathione redox (GR) cycle in cells exposed to high-glucose conditions using cultured human umbilical vein endothelial cells. Glutathione-dependent H2O2 degradation in cells exposed to 33 mmol/l glucose (HG) for 5-7 days was reduced by 48% vs. 5.5 mmol/l glucose (NG). This impairment under the oxidative stress was D-glucose-specific and concentration-dependent and was also associated with a 42% decrease in intracellular NADPH content. Exposure of cells to 200 micromol/l H2O2 stimulated the GR cycle and the pentose phosphate pathway (PPP) at the same time. In the HG condition, activation of PPP was reduced by 50%, which was consistent with a decrease in NADPH content. Inhibition of glycolysis by H2O2 was less marked in HG cells versus NG cells. Activation of polyol pathway in HG cells is not responsible for the decrease in intracellular NADPH content. These results indicate that activation of the PPP and NADPH supply to the GR cycle is impaired in HG cells exposed to H2O2, which may result in increased oxidative stress to endothelial cells.