Variation in the degree of prolonged (residual) biological activity of the melanotropin peptides alpha-MSH (alpha-melanocyte-stimulating hormone, Ac-Ser-Tyr-Met-Glu- His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and the superpotent analogues [Nle4,DPhe7]alpha-MSH (MT-I) and Ac-[Nle4,Asp5,DPhe7,Lys10]alpha-MSH(4-10-NH2 (MT-II) has stimulated considerable interest regarding this biological phenomena. We have examined the differences in their relative dissociation rates from the melanocortin receptor, hMC1R, to try and correlate peptide dissociation rates with the observations of prolonged biological activity. Interestingly, these studies revealed that alpha-MSH remained 25% bound, MT-I 65% bound, and MT-II 86% bound 6 h after the ligand had been removed from the assay medium. The relative dissociation rate of MT-II was 4 times slower than that for alpha-MSH and 2 times slower than that for MT-I, which was 2 times slower than that for alpha-MSH. These data suggest that slow dissociation kinetics (hours) may contribute to the prolonged biological activities observed for both MT-I and MT-II peptides in vitro and in vivo. The prolonged binding, biological activities, and enzymatic stability of MT-I and MT-II make them putative candidates for clinical uses such as external scintigraphy for the localization of tumors (i.e., melanoma).