Oxygen-derived free radicals are believed to be involved in diabetes-induced vascular complications. The role of oxygen radicals in endothelial dysfunction in diabetes is not known with certainty. In this study we tested whether inhibition of lipid peroxidation using the potent inhibitor U74389F, a 21-aminosteroid also known as lazaroid, could prevent endothelial dysfunction in diabetes. Lewis strain rats were made diabetic by intravenous injection of streptozotocin. A subgroup of diabetic animals received daily oral doses of 10 mg/kg U74389F at 72 hours post streptozotocin and throughout the 8-week duration of diabetes. Thoracic aortas were isolated and suspended in isolated tissue baths and contracted with norepinephrine. Relaxation due to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aorta while relaxation due to A23187 and nitroglycerin was unaltered. Chronic treatment of diabetic animals with U74389F normalized the increase in plasma lipid peroxides as assessed by thiobarbituric acid–reactive substances but did not alter serum insulin levels, blood glucose concentration, nor total glycosylated hemoglobin. Increases in aortic catalase activity resulting from diabetes was not altered by U74389F. Despite reductions in lipid peroxides, U74389F did not prevent the diabetes-induced impairment in endothelium-dependent relaxation caused by acetylcholine. These data suggest that other pathways that are antecedent to lipid peroxidation may be responsible for endothelial dysfunction in diabetes.