The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G- protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and α-, β-, and γ- melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent α-MSH analog [Nle4,D-Phe7]MSH (NDP-MSH) and the human MC1R we examined the effects of site-directed receptor mutagenesis on the binding affinity and potency of NDP-MSH. In addition, we also examined the effects of these same mutations on the binding affinity and potency of the structurally related agonists α- MSH, γ-MSH, and Ac-Nle4-cyclic-[Asp5,His6,D- Phe7,Arg8,Trp9,Lys10]NH2 (MT-II). Mutagenesis of acidic receptor residues Glu94 in TM2 and Asp117 or Asp121 in TM3 significantly altered the binding affinity and potency of all four agonists suggesting that these receptor residues are important to the ligand-receptor interactions of all. A disproportionate change in agonist potency versus affinity observed with simultaneous mutation of these acidic residues (mutant constructs D117A/D121A or E94A/D117A/D121A) or introduction of a single positive charge (mutant construct D121K) also implicates these residues in receptor activation. In addition, results from the individual mutation of aromatic receptor residues Phe175, Phe196, and Phe257, and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand- receptor interactions between these melanocortins and the hMC1R.