The relationship between melanogenesis and the expression of melanocyte stimulating hormone (MSH) receptors on the surface of melanocytes was examined using sublines generated from the melanotic JB/MS melanoma. JB/MS cells were propagated in long term culture to allow for phenotypic drift in their characteristics of differentiation, and then were cloned; the cloned cells ranged from well differentiated and pigmented to undifferentiated and amelanotic. Spontaneous and MSH-induced melanogenesis in these different lines was measured and correlated with the number of MSH receptors expressed. After 6 months of in vitro culture, the ability of the cells to respond to MSH was significantly reduced, as were the number of MSH receptors expressed; the cells had reduced pigmentation and were relatively undifferentiated histologically. Subsequently, clonally-derived pigmented cells were found to have numbers of surface MSH receptors (approximately 60,000 per cell) and levels of melanogenic activity similar to the original JB/MS cell line. However, an amelanotic clone had an even more dramatically reduced level of pigmentation which correlated with a further decrease in the expression of MSH receptors (less than 1,000 per cell) and the production of a potent melanogenic inhibitor. We also examined the responses of these various sublines to alpha, beta, and gamma-interferons and found significant heterogeneity in their abilities to respond to these cytokines. This study clearly shows that there is a direct correlation between melanogenesis and the expression of MSH receptors on the surface of melanocytes, and that melanogenic inhibitors may be critically involved in the regulation of mammalian pigmentation.