Bombesin receptor subtype-3 (BRS-3), a G-protein-coupled orphan receptor, shares 47% and 55% homology with other known mammalian bombesin receptors. Despite the molecular characterization of BRS-3, its function remains unclear as a consequence of its low affinity for bombesin and the absence of an identified natural ligand. Although the other mammalian bombesin receptors are widely distributed in the gut and central nervous system, expression of BRS-3 in the gastrointestinal tract has not been previously described. We report the expression of BRS-3 mRNA and protein in the tunica muscularis of the rat gastrointestinal tract. The mRNA expression pattern was studied by reverse transcription followed by quantitative polymerase chain reaction. To identify the cellular sites of expression of BRS-3, we performed immunocytochemistry by using a N-terminus-specific affinity-purified antiserum. BRS-3 was found to be widely expressed in the rat gastrointestinal tract at both the mRNA and protein levels. BRS-3-like immunoreactivity (BRS-3-LI) was localized in neurons of the myenteric and submucosal ganglia, being primarily concentrated near the neuronal plasma membrane, and in fibers distributed in the longitudinal and circular muscle layers. In addition, BRS-3-LI was observed in the cell bodies and processes of c-kit+ interstitial cells of Cajal. These data have functional applications for the effects mediated by the activation of BRS-3 on gut motility through distinct neuronal and non-neuronal pathways.