Recent evidence suggests that the melanogenesis intermediate 5-S-cysteinyldopa (5-S-CD) could display antioxidative activity. In the present study, the synthesis of 5-S-CD was examined in human epidermal melanocytes isolated from dark skin type VI (MT) and from white skin type III (GT). The MT melanocytes showed the higher melanin content and dopa oxidase activity. In addition, they produced eumelanin as shown by their ultrastructure, and the solubility and UV/visible absorption of the isolated pigment. Both MT and GT cells showed high levels of 5-S-CD (5.5-6.9 nmol/mg protein). 5-S-CD was also detected in culture supernatants from MT cells; the secretion rate was estimated to be 2.5 nmol/mg protein per 24 h. The role of cysteine and glutathione in 5-S-CD formation was investigated by exposing the melanocytes to the gamma-glutamylcysteine synthetase inhibitor L-buthionine sulfoximine (BSO). A strong reduction in glutathione levels (4-8% of the untreated controls) associated with an increase in cysteine levels (152-154%) was observed. In addition, BSO induced a moderate increase in the cellular levels of 5-S-CD (114-129%) and a decrease in dopa oxidase activity (75-83%). Our results indicate that the direct addition of cysteine to dopaquinone is the main source of 5-S-CD in human epidermal melanocytes. It is proposed that the synthesis of 5-S-CD is a mechanism regulating dopaquinone levels during pigment formation and/or a defence mechanism against oxidative stress.