Epitopes determining the agonist property of two structurally distinct selective ligands for the human bombesin receptor subtype 3 (BB3), [D-Tyr6,(R)-Apa11,Phe13, Nle14]-bombesin(6-14) (Pep-1) and Ac-Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2 (Pep-2), were mapped through systematic mutagenesis of the main ligand-binding pocket of the receptor. The mutational map for the smaller Pep-2 spanned the entire binding pocket of the BB3 receptor. In contrast, the much fewer mutational hits for the larger Pep-1 were confined to the center of the pocket, i.e., the opposing faces of the extracellular segments of transmembrane (TM)-III, TM-VI, and TM-VII. All the residues, which upon mutation affected Pep-1, were also hits for Pep-2 and included those that were most essential for the function of Pep-2: LeuIII:04 (Leu(123)), TyrVI:16 (Tyr(291)), and ArgVII:06 (Arg(316)). The BB3 receptor was found to signal with 12% ligand-independent activity that was strongly influenced both positively and negatively by several mutations in the binding pocket. The substitutions, which decreased the constitutive signaling, included not only the major mutational hits for the peptide agonists but also mutations more superficially located in the receptor. It is concluded that activation of the BB3 receptor is dependent upon an epitope in the main ligand-binding pocket at the interface between TM-III, TM-VI, and TM-VII that corresponds to the site where, for example, activating metal ion sites have been constructed previously in 7TM receptors.