The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe7 with D-Phe or D-naphthylalanine (D-Nal(2′)) in ACTH(1-24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe7 with D-Nal(2′) in ACTH(1-24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe7 with D-Phe7 in ACTH(1-17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in D-Phe ACTH binding affinity and potency. Our results suggest that Phe7 in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.