The oxidative half-reaction of xanthine oxidase is reexamined with regard to the generation of the superoxide anion. By using cytochrome c reduction to monitor superoxide, it is found that the stoichiometry of superoxide produced to enzyme reoxidized is 2:1, significantly greater than previously reported (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4350-4362). Furthermore, the kinetics of superoxide-dependent cytochrome c reduction exhibits a pronounced lag during the rapid phase of enzyme reoxidation and a limiting rate identical with that of the slow phase of enzyme reoxidation. This indicates that superoxide is generated only in the last steps of the sequential removal of reducing equivalents from the enzyme by molecular oxygen. Experiments with the two-electron-reduced enzyme indicate that it too produces two superoxide ions for each molecule of enzyme reoxidized, demonstrating that it is the last two electrons to leave the enzyme in the course of reoxidation that form superoxide. The sequential scheme for the oxidative half-reaction must therefore be 6 leads to 4 leads to 2 leads to 1 leads to 0, where the numbers reflect the number of reducing equivalents in each intermediate. Using this scheme, both enzyme reoxidation and cytochrome c reduction can be accurately simulated. Reasons for the different behavior of the two-electron-reduced xanthine oxidase compared to the six- and four-electron-reduced enzyme are discussed.