Four a-MSH drug conjugates have been synthesized, 2 Cterminal (Pep 3 and 4) and 2 central fragments (Pep I and 2), the latter being the 4-10 sequence known to be the main a-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locatiom. Their ability to recognize a-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep I and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled a-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptorrecognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicy in terms of lCw values, Pep I being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic t o all cells than melphalan itself. Furthermore, Pep I and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an a-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep I to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptormediated cytotoxicity mechanism occurring with aMSH central fragments in human melanoma cells due to the presence of a-MSH-specific receptors. This mechanism appeared to be both peptide-and cell-type-specific.