Three mammalian bombesin receptor subtypes have been characterized: the gastrin-releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). In a previous report we identified four amino acids that are critical for high affinity binding of bombesin and gastrin-releasing peptide (GRP) to the GRP-R. These four amino acids are conserved in all species variants of the GRP-R and NMB-R which bind bombesin with high affinity, but they are diverged in BRS-3, the bombesin receptor subtype that binds bombesin with much lower affinity. Substituting these four divergent amino acids in BRS-3 for the conserved amino acids in either GRP-R or NMB-R increased the affinity of the mutated BRS-3 (4DeltaBRS-3) for bombesin compared with wild-type BRS-3. We hypothesized that the same four amino acids might be critical for high affinity NMB binding to the NMB-R. In this study we confirm this hypothesis by showing that the affinity of NMB is increased in a mutant BRS-3 receptor (4DeltaBRS-3) that contains these four substitutions resulting in an affinity that is close to the affinity of wild-type NMB-R for NMB. In contrast, these four amino acid substitutions in BRS-3 did not result in the formation of a high affinity binding site for the recently described non-peptide NMB-R antagonist PD168368.