L-Gulono-gamma-lactone oxidase [EC 1.1.3.8] was purified 80-fold from rat liver microsomes. In confirmation of our previous finding with a cruder preparation, the purified enzyme was shown to contain an L-gulono-gamma-lactone-reducible pigment as a prosthetic group. This pigment was not liberated from the protein by acid ammonium sulfate, 10% trichloroacetic acid or 2 M area, but was effectively released by proteolytic digestion. The pigment thus released showed a reduced-minus-oxidized difference spectrum characteristic of a flavin compound. The pigment was liberated from a trichloroacetic acid-treated preparation of the enzyme by pronase digestion and purified by Florisil column chromatography and paper chromatography. The absorption spectrum as well as the fluorescence emission and excitation spectra of the purified pigment indicated that it was actually a flavin peptide. It was, however, different not only from FMN but also from flavin peptides isolated from other sources such as succinate dehydrogenase [EC 1.3.99.1] and monoamine oxidase [EC 1.4.3.4] as regards the pH dependence of fluorescence intensity and the Rf value on thin-layer chromatography. A preliminary analysis showed that the purified flavin compound contained several amino acid residues. Alkaline photolysis of the purified flavin peptide suggested that the isoalloxazine ring of the flavin is involved in its binding to the peptide. The hypsochromic shift of the absorption peak in the near-ultraviolet region suggested further that the linkage between the flavin and the peptide may be mediated by the 8-methyl group of the isoalloxazine nucleus. It can be concluded that the prosthetic group of gulonolactone oxidase is a flavin which is covalently bound to the enzyme protein.