Since the characterization of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as a major melanogenic intermediate, the fate of this compound and the mechanisms of its incorporation into the melanin polymer have become major issues in the study of melanogenesis. DHICA is a stable dihydroxyindole with a low rate of spontaneous oxidation, suggesting that enzymatic mechanism(s) might contribute to its evolution. The most obvious candidates are the melanosomal tyrosinases. We have recently shown that mouse melanosomes contain two electrophoretically distinct tyrosinase isoenzymes, termed low electrophoretic mobility tyrosinase (LEMT) and high electrophoretic mobility tyrosinase (HEMT), that can be resolved and purified. In this study, we report immunological evidence indicating that LEMT corresponds to the protein encoded by the brown locus (termed tyrosinase-related protein-1, TRP1), while HEMT corresponds to the tyrosinase encoded by the albino locus. We have compared the ability of both isoenzymes to catalyze DHICA evolution as determined by high performance liquid chromatography; although LEMT is a relatively poor tyrosine hydroxylase and DOPA oxidase as compared to HEMT, it was readily able to accelerate DHICA consumption concomitant with the production of a brownish product. However, the DHICA conversion activity of HEMT was barely detectable. The ability of purified LEMT to catalyze DHICA conversion could be almost completely abolished by treatment with heat or trypsin, and was inhibited in a concentration dependent way by the tyrosinase inhibitor 2-phenylthiourea and by L-tyrosine. Moreover, in the presence of low concentration of ascorbate, the DHICA conversion activity of LEMT displayed a lag period which was progressively longer at higher ascorbate concentrations. Based on the relationship between ascorbate added, enzyme activity, and lag period, it is very likely that the DHICA converting activity is indeed a DHICA oxidase activity. This was further proven by the demonstration that the product reacts rapidly and efficiently with the quinone trapping reagent 3-methyl-2-benzothiazolinone hydrazone, yielding a colored adduct similar to the one obtained with DOPAquinone. The DHICA oxidase activity of LEMT displayed a Km for DHICA of about 0.8 mM, as compared to 1.9 mM for L-DOPA and 0.23 nM for L-tyrosine. These results suggest that TRP1, the product of the brown locus, is indeed a tyrosinase with DHICA oxidase activity. However, as opposed to the tyrosinase encoded by the albino locus, TRP1's role in melanogenesis could be more directly related to DHICA metabolism than to the first steps of the pathway.