Ultraviolet radiation (UVR), the synthetic diacyglycerol (DAG), 1-oleoyl-2-acetylglycerol (OAG), and cyclic AMP (cAMP) stimulants, including cholera toxin (CT) have all been shown to increase melanogenesis in cultured human melanocytes. Indirect evidence suggests that an increase in intracellular free Ca2+ ([Ca2+]i) may be important in stimulated melanogenesis. Therefore, to determine whether melanogenic responses are modulated by [Ca2+]i, the Ca2+ in the culture medium of melanocytes ([Ca2+]o) was raised from 70 microM to 1 mM. This switch in [Ca2+]o was associated with a biphasic increase in [Ca2+]i, with an early transient rise, over minutes, and a delayed sustained rise in [Ca2+]i, over hours. The early increase was blocked by nickel chloride (NiCl2), but not affected by depletion of [Ca2+]i stores by thapsigargin, suggesting that this [Ca2+]i rise was due to Ca2+ entry across the plasma membrane. Melanocytes cultured in the absence of CT had a reduced basal melanin content following the switch to 1 mM [Ca2+]o, but in the presence of CT, which acts by stimulating cAMP synthesis, the basal level was increased. Raising [Ca2+]o resulted in enhanced melanogenic responses to UVR and OAG, in the presence or absence of CT, suggesting that Ca(2+)-dependent mechanisms are important. UVR also stimulated a delayed rise in [Ca2+]i, over 24 h, but OAG did not. These results indicate that while [Ca2+]i is not essential for melanogenesis, it plays an important role in modulating the responses of melanocytes to melanogenic stimuli.