In cultured cells of the Bomirski Ab amelanotic hamster melanoma line, the substrates of tyrosinase, L-tyrosine, and L-DOPA induce the melanogenic pathway. In this report, we demonstrate that these substrates regulate the subcellular apparatus involved in their own metabolism and that this regulation is under the dynamic control of one of the components of this apparatus, tyrosinase, via tyrosine hydroxylase activity. Culturing cells with nontoxic but melanogenically inhibitory levels of phenylthiourea (PTU; 100 microM) strongly inhibits induction of both the tyrosine hydroxylase and DOPA oxidase activities of tyrosinase by L-tyrosine (200 microM) but has no effect on the induction of either activity by L-DOPA (50 microM). De novo synthesis of premelanosomes precedes the onset of tyrosine-induced melanogenesis. Thereafter, increases in the population of melanosomes (likewise inhibited by PTU) correlate positively with increases in tyrosinase activity induced by L-tyrosine. Melanogenesis induced by L-DOPA in the absence of L-tyrosine is rate-limited not by tyrosinase but by inadequate melanosome synthesis. Our findings indicate that in Bomirski Ab amelanotic hamster melanoma cells the synthesis of the subcellular apparatus of melanogenesis is initiated by L-tyrosine and is regulated further by tyrosinase and L-DOPA, which serves as a second messenger subsequent to tyrosine hydroxylase activity.