Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme.