The melanocortin receptor (MCR) subtype family is a member of theGPCRsuperfamily, and each of them has a different pharmacological profile with regard to the relative potency of the endogenous and synthetic melanocortin peptides. α-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R, and MC5R. In this study, we examined the role of Phe7 in ACTH on human (h) MC1R, MC3R, and MC4R binding and signaling. Our results indicate that substitution of Phe7 with D-Nal(2′)7 in ACTH1-24 yields a pharmacological profile different from that for substitution of Phe7 with D-Nal(2′)7 in MSH in hMC1R, hMC3R, and hMC4R. N-D-Nal(2′)7-ACTH1-24 is an agonist at hMC3R and hMC4R which did not change the peptide from an agonist to an antagonist at hMC3R and hMC4R. Further experiments indicate that N-D-Nal(2′) 7-ACTH1-17 is the minimal peptide required for hMC3Rand hMC4R activation. Single-amino acid substitution studies of D-Nal(2′) 7-ACTH1-17 indicate that amino acid residues 15-17 in N-D-Nal(2′)7-ACTH1-17 are crucial for hMC3R and hMC4R activation. Substitutions of these amino acid residues reduced or abolished agonist activity at hMC3R and hMC4R. Conformational studies revealed a new β-turn (Arg8-Trp9-Gly10-Lys11) in N-D-Nal(2′)7-ACTH1-17, compared to the β-turn-like structure at NDP-α-MSH (His6-D-Phe7-Arg 8-Trp9). Our results suggest that NDP-α-MSH and N-D-Nal(2′)7-ACTH1-17 do not share the same binding site; the highly basic C-terminal fragment (Lys15-Lys16-Arg 17) of N-D-Nal(2′)7-ACTH1-17 induced a new β-turn, and this shift contributed the selective agonist activity at hMC3R and hMC4R.