In studies to determine whether pigmentation can be regulated physiologically by thiols, human melanoma cells (MM418c5) and melanocytes were found to become depigmented when cultured continuously in 50 microM cystamine. Cystamine was depleted from the culture medium and the treatment was nontoxic and reversible. Cysteamine, dithiothreitol, and phenylthiourea were less effective, and glutathione, cysteine, and cystine were inactive. Tyrosinase (dopa oxidase) activity was not greatly affected except for induction of a lag period. In contrast, tyrosinase activity in an amelanotic melanoma cell line (MM96L) was rapidly inhibited without consumption of cystamine/cysteamine, in association with the generation of free thiol in the culture medium, and could be enhanced by the cystine transport inhibitor, glutamate. Tyrosinase expressed by a recombinant vaccinia virus was inhibited by cystamine treatment of MM96L and HeLa cells. Cystamine treatment lowered the degree of cross-linking of the pigmentation antigen gp75/TRP-1 in MM418c5 cells. Tyrosinase protein and mRNA levels in MM418c5 cells were not affected by cystamine. The results show that cystamine at a concentration close to physiologic levels has multiple effects on the melanogenic pathway. In amelanotic cells, tyrosinase has a short half-life and is readily inhibited by cystamine/cysteamine whereas tyrosinase in the more mature melanosomes of the pigmented cell appears to be less accessible to proteolytic and thiol attack. Inhibition of melanin synthesis in the latter cell type may arise to a significant degree from reduction of cystamine to cysteamine, which sequesters quinones.