We have introduced topographical constraints at the 9 position of a superpotent cyclic alpha-melanotropin analogue, Ac-Nle4-Asp5-His6-DPhe7-Arg8-Trp9-Lys10-NH2, by incorporating a methyl group at the beta-carbon of Trp9. These studies were performed on the Trp side chain pharmacophore to identify the bioactive topography of the indole moiety with melanocortin MC1 receptors. The four beta-MeTrp9 isomers, in addition to the stereochemical controls L- and DTrp9, were used to probe differential receptor molecular recognition of the tryptophan moiety in two bioassay systems. Approximately a 460-fold difference in potency was observed between the diastereoisomeric peptides in the frog skin bioassay, with only 33- and 10-fold efficacy differences observed in binding and intracellular cAMP accumulation, respectively, on the human melanocortin receptor, hMC1R. The relative orders of potencies in the frog skin bioassay were 2R,3S > 2S,3S = 2R,3R >> 2S,3R and for the hMC1R were 2S,3S > 2R,3R > 2R,3S >> 2S,3R. Of particular interest is the ability of these topographically constrained ligands to differentially affect prolonged biological activity. The 2R,3R diastereoisomeric peptide possessed superprolonged activity, whereas the 2S,3S peptide lacked any residual activity in the frog skin bioassay. However, on the melanocortin receptor, the 2S,3S diastereoisomeric peptide maintained slow dissociation rates (t1/2 = 7 h), while the other diastereoisomeric peptides possessed dissociation t1/2 rates of ca. 2 h. These data strongly implicate ligand-receptor interactions and kinetics as contributing to the observed prolonged biological activities and clearly illustrate topographical recognition differences between these two peripheral MC1 receptors involved in skin pigmentation. This study also demonstrates that topographical modifications of pharmacophore side chain residues, in addition to identifying preferential side chain orientation, can be a useful strategy for the design of peptides to increase the duration of biological activity, relative to the native ligand.