In mammals, melanin exists in two chemically distinct forms: the red-yellow phaeomelanin and the brown-black eumelanin. Although administration of the pigmentary hormone alpha-melanocyte-stimulating hormone (alpha MSH) and its synthetic analogue Nle4DPhe7 alpha MSH induces skin darkening in man, the increases in melanogenesis in cultured human melanocytes in response to these peptides are relatively small. However, it is possible that MSH affects the eumelanin:phaeomelanin ratio rather than total cellular melanin. Thus, this study examined the specific effects of Nle4DPhe7 alpha MSH on the two melanins in cultured human melanocytes, quantifying eumelanin and phaeomelanin by high performance liquid chromatography. Nle4DPhe7 alpha MSH induced significant increases in the eumelanin content of these cells while having lesser and varied effects on the levels of phaeomelanin. As a consequence, the eumelanin:phaoemelanin ratio was increased in every culture. These results demonstrate that Nle4DPhe7 alpha MSH affects melanin type in human melanocytes and suggest a possible mechanism by which this peptide induces skin darkening in man.