Tyrosinase, the critical enzyme to melanin pigmentation in mammals, occurs as a series of isozymic forms, which have been previously regarded as different stages in processing of a single precursor form. Recently, three different cDNA clones have been identified which may encode tyrosinase, they share extensive sequence homology but are distinct; two of them have been mapped to genetic loci which regulate different aspects of melanogenesis. Since direct confirmation of the authentic tyrosinase sequence has proven impossible by conventional protein sequencing strategies, we have approached the identification of the tyrosinase gene by synthesizing peptides encoded by the putative genes and preparing antibodies to those peptides. By use of pulse-chase labeling and immunoprecipitation analyses, and by enzymatic determinations, pMT4 (which maps to the brown b locus in mice) is shown to encode a molecule with tyrosinase catalytic activity which is biochemically identical with authentic tyrosinase. However, our results raise the possibility that other gene products may contribute to melanogenesis by one or more melanogenic activities.