In animals, the coat-darkening effects of alpha-melanocyte stimulating hormone (alpha-MSH) are opposed by agouti protein. Although agouti protein has been shown to be a competitive antagonist of the melanocyte-associated MC-1 melanocortin receptor, the possibility that agouti protein can affect melanogenesis independently of its ability to antagonise melanocortin activity cannot be excluded. This study demonstrates that murine agouti protein causes both a time- and concentration-dependent suppression of melanogenesis in B16 F1 murine melanoma cells. In addition, human agouti protein decreases melanogenesis in cultured human epidermal melanocytes. However, agouti protein has little effect on the ability of alpha-MSH to stimulate melanogenesis. These observations raise fundamental questions about the mode of action of agouti protein in regulating melanogenesis.