Superoxide anion (O2-) blocks the vascular effect of endothelium-derived relaxing factor (EDRF). Previous reports implicate L-arginine or N alpha-substituted arginine compounds as precursors of EDRF. Employing a microassay for the measurement of O2- production by rabbit aortic rings, which is based on the established method of reduction of cytochrome c by O2-, we studied the interaction between O2- and EDRF using L-arginine, an N-substituted arginine compound, namely, N alpha-benzoyl-L-arginine ethyl ester (BAEE), sodium nitroprusside and NG-monomethyl-L-arginine (NMMA), a putative specific inhibitor of EDRF synthesis. Measurements of O2- production were made under basal conditions and upon stimulation with alloxan, the diabetogenic, O(2-)-generating compound. Both BAEE, an EDRF-generating agent (0.3 and 3.0 mM) and sodium nitroprusside, an NO-generating compound (1.7 and 3.4 microM), significantly reduced alloxan-stimulated O2- production, but L-arginine (0.6-3.0 mM) paradoxically increased O2- generation. NMMA (1.0 mM) blocked the inhibitory effect of BAEE on O2- production in an endothelium-dependent manner. Because NMMA is an inhibitor of EDRF, these results suggest that BAEE, but not L-arginine, reduces O2- produced by the endothelium of the rabbit aorta through a mechanism involving EDRF generation.