Antagonist ligands of the melanocortin-3 and -4 receptors (MC3R, MC4R), including agouti-related protein (AGRP), are postulated to be targets for the treatment of diseases of negative energy balance. Previous studies reported the macrocyclic MC3R/MC4R antagonist c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-dPro8], which is 250-fold less potent at the mouse (m) mMC3R and 3-fold less potent at the mMC4R than AGRP. Previous studies explored the structure-activity relationships around individual positions in this template. Herein, a multiresidue substitution strategy is utilized, combining the lead sequence with hPhe4, Dap5, Arg5, Ser6, and Nle7 substitutions previously reported. Two compounds from this study (16, 20) contain an hPhe4/Ser6/Nle7 substitution pattern, are 3-6-fold more potent than AGRP at the mMC4R and are 600-800-fold selective for the mMC4R over the mMC3R. Another lead compound (21), possessing the hPhe4/Arg5 substitutions, is only 5-fold less potent than AGRP at the mMC3R and is equipotent to AGRP at the mMC4R.