The electron transfer mechanism in the reduction of aromatic nitro compounds by xanthine oxidase was investigated using methyl p-nitrobenzoate and p-nitroacetophenone as substrates. Methyl p-nitrobenzene was reduced by both one-electron and more than two-electron transfer mechanisms in the enzyme-electron donor system. When NADH was used as an electron donor, the ratio of one-electron flux to the total electron flux (the summation of one-electron and more than two-electron fluxes) was dependent on pH of the medium, but not on the concentration of the nitro compounds. The reverse was the case when the electron donor was xanthine. Additional experiments showed that methyl p-nitrobenzoate or p-nitroacetophenone was reduced to the corresponding hydroxylamino compounds and amino compounds by xanthine oxidase supplemented with xanthine or NADH. In these cases, the pattern of formation of the reduction products was dependent on the enzyme activity. The present study strongly suggested that the reduction of aromatic nitro compounds by xanthine oxidase proceeds through the four-electron and six-electron transfer mechanisms as well as the one-electron transfer mechanism.