We characterized the bombesin receptor population in the rat stomach and determined the receptor subtype mediating the contractile effect of bombesin in the gastric fundus. Using in vitro receptor autoradiography, we evaluated the ability of the specific gastrin-releasing peptide-preferring receptor antagonist [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester to inhibit binding of 125I-[Tyr4]bombesin to the gastric fundus, corpus and antrum. Binding to these regions was completely inhibited by [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester suggesting that these receptors are the gastrin-releasing peptide-preferring subtype. We found that the rank order of potency for the contractile effect of bombesin, and the related mammalian peptides neuromedin C and neuromedin B, was bombesin > neuromedin C > neuromedin B. [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester was equipotent in antagonizing contractions produced by all three peptides. Furthermore, receptor tachyphylaxis to either neuromedin C or neuromedin B abolished the subsequent contractile response elicited by neuromedin C and neuromedin B, suggesting that one bombesin receptor subtype mediates rat gastric fundal contractions. Together, these results demonstrate that the bombesin receptor subtype in the rat stomach is gastrin-releasing peptide-preferring subtype and that this subtype is responsible for the effects of bombesin-like peptides on fundal smooth muscle contraction.