We describe results demonstrating the positive regulation of melanogenesis by two substrates of the melanogenic pathway. We have found that L-tyrosine and L-dihydroxyphenylalanine (L-dopa), whose metabolic fates are affected by the activity of that pathway, can also act as its regulators. In living pigment cells, tyrosinase (EC 18.104.22.168), a crucial and rate-limiting enzyme of melanogenesis, acts in subcellular organelles known as melanosomes. Melanin is laid down only in these organelles. We demonstrate that supplementing Ham's F-10 medium with additional L-tyrosine or L-dopa during the culture of amelanotic Bomirski hamster melanoma cells results in a rapid increase in melanin formation, which is not simply due to greater availability of substrate. There is a rapid increase in tyrosinase activity and a large scale synthesis of melanosomes. The effects of L-tyrosine and L-dopa are prevented by the addition of cycloheximide. The actions of L-tyrosine and L-dopa are specific in that under similar conditions D-tyrosine, D-dopa, N-acetyl-L-tyrosine, L-phenylalanine, L-tryptophan and L-valine have little or no effect. The two substrates, L-tyrosine and L-dopa, appear to act through related but distinct mechanisms. Our findings provide an example of a little-known phenomenon: regulation of a differentiated eukaryotic phenotype through positive control by substrates in the pathway.