The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/P melanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/P melanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes. J. Cell. Physiol. 191: 162–172, 2002. © 2002 Wiley-Liss, Inc.