Six tyrosinase isozymes were purified from the browned gill of the fruiting body of Lentinus edodes by ammonium sulfate fractionation, DEAE-Sephacel and Q-Sepharose column chromatography, and partially denaturing SDS-PAGE. At the step of Q-Sepharose column chromatography, two active fractions (A and B) were obtained. Each fraction was separated to three further fractions, A1, A2, and A3, and B1, B2, and B3, respectively, by partially denaturing SDS-PAGE. All these isozymes consisted of two types of polypeptides: alpha polypeptide (A alpha or B alpha) and either beta (A beta or B beta) or gamma polypeptide (A gamma or B gamma). The alpha polypeptide contained the consensus amino acid sequence of the active site of known tyrosinases, which is considered to act as a catalytic subunit. From the results of peptide mapping and the amino acid composition, A alpha and B alpha polypeptides were considered to be different proteins. The kinetic properties of the purified tyrosinase isozymes differed greatly according to whether they contained beta or gamma polypeptide, indicating these polypeptides to be a possible regulatory subunit.