Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane alpha-helices (TM) 2, 3, 6, and 7 (residues Glu(100), Asp(122), Asp(126), Phe(261), His(264), Leu(265), and Leu(288)). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor-ligand complexes with their agonists were generated by distance-geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys(40)-Cys(279), Cys(271)-Cys(277)) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His(6), d-Phe(7), Arg(8), and Trp(9)) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His(6) and Arg(6) of NDP-MSH; their substituted piperidines mimic Trp(9) of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu(133), Trp(258), and Phe(261) residues.