Bombesin receptor subtype (BRS)-3, a G-protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin (Bn) receptor for gastrin-releasing peptide. There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control, motility, and tumor growth. BRS-3 has low affinity for all Bn-related peptides; however, recently synthetic high-affinity agonists, [d-Tyr(6)/d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14), were described, but they are nonselective for BRS-3 over other Bn receptors. Based on these peptides, three BRS-3-selective ligands were developed: peptide 2, [d-Tyr(6)(R)-3-amino-propionic acid(11),Phe(13),Nle(14)]Bn(6-14); peptide 3, [d-Tyr(6),(R)-Apa(11),4Cl-Phe(13),Nle(14)]Bn(6-14); and peptide 4, acetyl-Phe-Trp-Ala-His-(tBzl)-piperidine-3 carboxylic acid-Gly-Arg-NH(2). Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this, we used a chimeric/site mutagenesis approach. Substitution of extracellular domain 2 (EC2) of BRS-3 by the comparable gastrin-releasing peptide receptor (GRPR) domain decreased 26-, 4-, and 0-fold affinity for peptides 4, 3, and 2. Substitution of EC3 decreased affinity 4-, 11-, and 0-fold affinity for peptides 2 to 4. Ten-point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107 (EC2-BRS-3) for lysine (H107K) (EC2-GRPR) decreased affinity (25- and 0-fold) for peptides 4 and 1; however, it could not be activated by either peptide. Its combination with Val101 (TM2), Gly112 (EC2), and Arg127 (TM3) resulted in complete loss-of-affinity of peptide 4. Receptor-modeling showed that each of these residues face inward and are within 4 A of the binding pocket. These results demonstrate that Val101, His107, Gly112, and Arg127 in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3 selectivity of peptide 4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/receptor interactions with peptide 4 are critical for both binding and activation. Furthermore, these result demonstrate that even though these three BRS-3-selective agonists were developed from the same template peptide, [d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14), their molecular determinants of selectivity/high affinity varied considerably.