Oxygen radicals are believed to play a role in vascular complications of diabetes mellitus. In this study, we evaluated whether long-term treatment with an iron chelator and inhibitor of metal-catalyzed hydroxyl radicals (.OH) could prevent diabetes-induced defects in endothelium-dependent relaxation. Diabetes was induced in Sprague-Dawley rats by injection of streptozotocin. At 48 h after streptozotocin, a subgroup of diabetic rats received daily injections of 50 mg/kg hydroxyethyl starch conjugated-deferoxamine (HES-DFO) for a total of 8 weeks. Long-term treatment with HES-DFO did not modify serum insulin or blood glucose taken at the end of the study; however, a modest reduction in glycosylated hemoglobin was present. In precontracted aortic rings suspended in tissue baths, endothelium-dependent relaxation to acetylcholine was impaired in diabetic rings compared with control rings in the presence or absence of indomethacin. Endothelium-independent relaxation to nitroglycerin was unaltered. Long-term treatment with HES-DFO had no effect on relaxation to nitroglycerin but completely prevented the impaired relaxation to acetylcholine in diabetic rings in either the presence or absence of indomethacin. These data suggest that iron-catalyzed .OH formation contributes to the development of diabetes-associated endothelial dysfunction.