The substrates and intermediates involved in the conversion of tyrosine or 3,4-dihydroxyphenylalanine into melanin by autooxidation, or tyrosinases (monophenol, dihydroxyphenylalanine:oxygen oxidoreductases, EC 1.14.18.1) of mushroom or mammalian melanocyte origin, was studied by a variety of enzymic assays, and by amino acid analysis. It was found that the classic pathway of melanin formation was followed, and that the proposed alternate pathway involving formation of the intermediate 3,4,6-trihydroxyphenylalanine was not a functional route, since nascent trihydroxyphenylalanine was not detectable. The ability of isolated mammalian tyrosinases to convert tyrosine into dihydroxyphenylalanine was unequivocably demonstrated. The polymerization of monomers into melanin was followed by the use of specifically labelled precursors, and the data indicate that uncyclized and carboxylated derivatives are not incorporated into the polymer in vitro. It was found that although in most respects the melanin produced from tyrosine by mushroom and mammalian tyrosinses are similar, the control mechanisms involved in the expression of melanin formation in these organisms must differ greatly.