(1) Bombesin (BN), originally isolated from amphibians, is structurally related to a family of BN-like peptides found in mammals, which include gastrin-releasing peptide (GRP) and neuromedin B (NMB). These peptides have important effects on secretion, smooth muscle contraction, metabolism and behavior. Here we report cloning and characterization of two subtypes of BN-like peptide receptors in Aves. (2) The amino-acid sequence of chick GRP-R (chGRP-R) is highly identical with mammalian and amphibian GRP-R, and this receptor showed high affinity for GRP, BN and synthetic bombesin agonist, [D-Phe(6), beta-Ala(11), Phe(13), Nle(14)]bombesin(6-14) ([FAFNl]BN(6-14)). The chGRP-R gene was localized to chicken chromosome 1q23distal-q24proximal, where chick homologs of other human X-linked genes have also been mapped. (3) ChBRS-3.5, having sequence similarities to both mammalian bombesin-like peptide receptor subtype-3 and amphibian bombesin-like peptide receptor subtype-4, showed high affinity for [FAFNl]BN(6-14), moderate affinity for BN, but low affinity for both GRP and NMB. (4) Expression of both receptors was detected in brain, but only chGRP-R was expressed in gastrointestinal (GI) tissues. (5) When expressed in Chinese hamster ovary K1 cells, these receptors mediate intracellular calcium mobilization upon agonist stimulation. These results suggest that a novel BN peptide may occur in Aves as an endogenous ligand for chBRS-3.5. (6) The receptor sequences responsible for ligand selectivities were discussed and this knowledge about avian BN-like peptide receptors will help us to understand the molecular basis for agonist sensitivities of BN-like peptide receptors.