The catechol 5-S-cysteinyldopa (5-S-CD) is produced in large amounts in metastatic malignant melanoma. To further understand the mechanism of formation of 5-S-CD, we investigated the effects of thiol modulating agents and melanin precursors on human melanoma cells. Under standard culture conditions (0.1 mM cystine), the cell levels of 5-S-CD were highly correlated with the degree of melanization and the dopa oxidase activity of the four cell lines investigated (Me8, JUSO, GLL19, Swift). Inhibition of glutathione (GSH) biosynthesis with buthionine sulphoximine did not affect 5-S-CD levels in the low melanotic GL 19 cells. In contrast, the highly pigmented Swift cells showed a strong increase in the cell levels of cystine (CysH) and 5-S-CD. When the cystine concentration of the growth medium was increased to 0.2 mM, a similar situation of 5-S-CD synthesis caused by an increase in intracellular CysH levels was observed in the Swift cells. The GLL19 cells showed enhanced 5-S-CD formation in the presence of 0.1 mM L-dopa. This effect was associated with a fourfold increase in dopa oxidase activity. Our data clearly indicate that 5-S-CD is formed in human melanoma cells by a tyrosinase-dependent mechanism involving the addition of CysH to dopaquinone. Based on the enhancing effect of buthionine sulphoximine on 5-S-CD formation, it is proposed that GSH is not directly implicated in 5-S-CD formation, but regulates CysH levels via the enzyme gamma-glutamylcysteine synthetase.