In the present study, the role of reactive oxygen species and the contribution of antioxidant defence in the time course of changes in acetylcholine-stimulated endothelium-dependent and sodium nitroprusside-stimulated endothelium-independent relaxation were investigated in aortic rings isolated from 6-month streptozotocin-diabetic and age-matched control rats. Although there were no significant differences in the degree of the peak relaxations produced by a single administration of acetylcholine (1 microM) or sodium nitroprusside (0.01 microM) between control and diabetic rings, the endothelium-dependent and -independent relaxant responses were more transient and the time required to reach a peak relaxation after addition of acetylcholine was shorter in diabetic vessels. Pretreatment of diabetic vessels with superoxide dismutase (100 U/ml) normalized the recovery phases of endothelium-dependent and -independent relaxations, but had no effect on the peak responses to acetylcholine and sodium nitroprusside. In the presence of diethyldithiocarbamate (5 mM), an inhibitor of superoxide dismutase, the transient nature of the relaxant response to acetylcholine or sodium nitroprusside was more marked and the peak relaxations were inhibited; these effects of diethyldithiocarbamate were more pronounced in diabetic than in control rings. Catalase, 160 U/ml, decreased the peak relaxant response to acetylcholine and accelerated fading of the relaxation in diabetic aorta. Similar results were obtained for control aorta with a higher concentration of catalase (550 U/ml). Pretreatment with 3-amino-1,2,4 triazole (5 mM), a catalase inhibitor, inhibited the peak relaxant response to acetylcholine in diabetic rings. The combination of superoxide dismutase (100 U/ml) plus 3-amino-1,2,4 triazole (5 mM) produced an increase of the transient nature of endothelium-dependent relaxation of diabetic rings greater than that with 3-amino-1,2,4 triazole alone. Neither catalase nor 3-amino-1,2,4 triazole affected the characteristics of sodium nitroprusside-induced relaxation. Desferrioxamine, an inhibitor of hydroxyl radical (.OH) production, or mannitol, a.OH scavenger, had no effect on the characteristics of either acetylcholine- or sodium nitroprusside-induced relaxation in control and diabetic rings. Biochemical measurements revealed an inhibited superoxide dismutase activity in diabetic aorta together with activated catalase. Our findings suggest that, during the chronic phase of streptozotocin-diabetes, excess superoxide (O(2)(. -)) is responsible for the enhanced transient nature of endothelium-dependent and -independent relaxation of aorta via a reduction in bioavailable concentrations of nitric oxide (NO). However, the involvement of hydrogen peroxide (H(2)O(2)) in the establishment of acetylcholine-stimulated relaxation may be increased, which is likely to account for the maintenance of the relaxant effect of acetylcholine in chronically diabetic vessels.