The reduction of milk xanthine dehydrogenase by salicylate anion radical (SL-), nicotinamide adenine dinucleotide radical (NAD.), and 1-methylnicotinamide (NMA) radicals was investigated by the use of pulse radiolysis. Reduction of the dehydrogenase with SL- proceeded via two phases. From the kinetic difference spectra obtained, the faster and slower phases of reduction represent that of one of the iron-sulfur centers and of FAD, respectively. The rate constant of the faster phase increased with the concentration of the enzyme, suggesting that the reduction follows a bimolecular reaction of SL- with the iron-sulfur center. In contrast, the rate constant of the slower phase (510 s-1) was independent of the concentration of the enzyme at pH 7.5. In order to elucidate the contribution of the molybdenum site in the reaction, a similar reaction was performed with enzyme modified with oxipurinol. In the modified enzyme, the slower phase was lost, whereas the faster phase was not affected. These results suggest that the slower phase is due to intramolecular electron transfer from the molybdenum center to FAD. On the other hand, NAD. reacted predominantly with FAD of the dehydrogenase to form the neutral semiquinone of FAD with a second order rate constant of 1.4 x 10(7) M-1 s-1 at pH 7.5, whereas a similar reaction in the oxidase, which was converted from xanthine dehydrogenase by proteolytical cleavage, was not observed. This suggests that NAD. transfers an electron via the binding site for NAD+ on the dehydrogenase. In contrast, NMA radical reduced only an iron-sulfur center of the dehydrogenase with a second order rate constant of 6.5 x 10(7) M-1 s-1 at pH 7.5.