Bombesin, a bioactive peptide first identified in amphibian skin, is related to two mammalian peptides, gastrin-releasing peptide (GRP) and neuromedin B. A family of 3 GPCRs, including GRP-R (BB1), NMB-R (BB2) and BRS-3 (BB3), mediate the biological effects of the peptides (Ohki-Hamazaki et al., 2005). BB3 differs from the others by its low affinity for bombesin. Although an endogenous ligand for BB3 has yet to be identified, a synthetic nonselective bombesin-like peptide [H-D-Phe6, β-Ala11, Phe13, Nle14]-bombesin-(6-14)-nonapeptide amide (Bombesin (6-14) Analog) activates BB3 with high potency. BB3-null mice have an obese phenotype (Matsumoto and Iijima, 2003). BB3 membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of antagonists of BB3 receptor interactions with its ligand. The membrane preparations exhibit a Kd of 0.23 nM for [125I]-Bombesin (6-14) Analog. With 10 mg/well of BB3 Membrane Prep and 0.3 nM [125I]-Bombesin (6-14) Analog, a greater than 3-fold signal-to-background ratio was obtained.