As an initial characterization of bombesin binding sites in the rat central nervous system, we examined the pharmacological specificity of binding of bombesin (BBS) and several BBS analogs to rat cortex and compared these results to those for rat pancreas. In addition, we used in vitro receptor autoradiography to evaluate binding of 125I-[Tyr4]bombesin and 125I-Bolton-Hunter neuromedin B (NMB) to several regions of the rat brain. The results of the pharmacological study indicated that the pancreas and cortex had different binding affinities for BBS-like peptides. While cortical binding sites had a high affinity for NMB, pancreatic binding sites had almost no affinity for NMB. Results from the autoradiographic study demonstrated that BBS receptor heterogeneity exists in individual nuclei in the rat brain. Some nuclei have a high affinity for NMB, similar to cortical BBS binding sites, other regions have a low affinity similar to pancreatic BBS binding sites. These results provide evidence that subtypes of BBS receptors are present in different tissues and within discrete regions of the rat central nervous system.