Although insulin-resistant states have been associated with endothelial dysfunction due to increased vascular oxidative stress, the underlying mechanisms are pooly understood. Recent experimental evidence suggests that tetrahydrobiopterin (BH₄), the natural and essential cofactor of NO synthases (NOS), plays a crucial role not only in increasing the rate of NO generation by NOS but also in controlling the formation of superoxide anion (O₂⁻) in endothelial cells. Because insulin resistance has been suggested to be a significant contributing factor in the development of abnormal pteridine metabolism and endothelial dysfunction, we investivated pteridine content and NO/O₂⁻ production with the use of isolated thoracic aortas obtained from fructose-induced insulin-resistant rats. Under insulin-resistant conditions where BH₄ levels are suboptimal, the production of O₂⁻ by NOS leads to endothelial dysfunction. Furthermore, oral supplementation of BH₄ restores endothelial function and relieved oxidative tissue damage, at least in part, through activation of endothelial NOS (eNOS) in the aorta of insulin-resistant rats. These results indicate that insulin resistance may be a pathogenic factor for endothelial dysfunction through impaired eNOS activity and increased oxidative breakdown of NO due to enhanced formation of O₂⁻, which are caused by relative deficiency of BH₄ in vascular endothelial cells.