Endothelium-derived relaxing factor (EDRF) is rapidly inactivated by radicals. Endothelial cells possess several antioxidant defense mechanisms. It is not clear which intrinsic antioxidant defense systems are important to preserve the release of biologically active EDRF. We impaired antioxidant defense in normal vascular tissue by inhibiting catalase activity with 3-amino-1,2,4-triazole (AT), superoxide dismutase with diethyldithiocarbamate (DETC), and by reducing glutathione content via inhibiting glutathione synthesis with L-buthionine-(S,R)-sulfoximine (BSO). Pretreatment of rabbit aorta in vitro with DETC markedly reduced endothelium-dependent relaxation in response to acetylcholine and calcium ionophore A23187 and, to a lesser extent, reduced endothelium-independent relaxation in response to nitroprusside. Pretreatment of cultured bovine aortic endothelial cells (BAEC) with DETC did not alter release of nitrogen oxides (measured by chemiluminescence), but, the effluent of pretreated cells showed marked depression in vasodilator activity (measured by bioassay). Pretreatment of rabbit aorta in vitro with AT did not alter endothelium-dependent and -independent relaxations. Pretreatment of BAEC with BSO did not alter the release of nitrogen oxides or the vasodilator activity. These results suggest that endothelial superoxide dismutase activity, but not catalase or glutathione, is necessary for the release of biologically active EDRF. An imbalance of the intrinsic superoxide dismutase and the production of superoxide anions may therefore predispose to impaired endothelium-dependent relaxations and alter vascular reactivity.