Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae. This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction. Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin. The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein. The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH. These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation. The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate. 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0. In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride. Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.