Previously we have found that binding of the nonpeptide substance P antagonist, CP 96,345, to the neurokinin-1 (NK-1) receptor was critically dependent on two short segments adjacent to the top of transmembrane segments (TM) V and VI, called segments A (residues 183-195) and D (residues 271-276), respectively. In the present study we have systematically performed substitutions of nonconserved residues within these two segments with residues from the homologous NK-3 and/or NK-2 receptor. In segment A, deletion of residues Glu193 and Lys194, which are not present in the NK-3 receptor, or substituting them with leucines as in the NK-2 receptor, decreased the affinity of CP 96,345 10- and 22-fold, respectively. Surprisingly, switching the position of Glu193 and Lys194 did not affect the affinity of CP 96,345, suggesting that, rather than interacting directly with CP 96,345, an interaction of these residues with one another is important for CP 96,345 binding. In segment D substitution of Tyr272 with threonine as in the NK-2 receptor and with alanine as in the NK-3 receptor decreased the affinity of CP 96,345 7- and 24-fold, respectively. Mutation of the preceding Pro271 to glycine alone did not affect CP 96,345 binding, but, combined with the mutation of Tyr272 to threonine, the affinity decreased 28-fold. A series of CP 96,345 analogues with modifications of the major chemical moieties exhibited equally reduced affinity as that of CP 96,345 for the Tyr272- and Lys193-Glu194-substituted constructs, except CP 95,555, which lacks one of the phenyl rings in the benzhydryl group and which was almost unaffected by these mutations. In conclusion, our data indicate a direct interaction between CP 96,345 and Tyr272, which are located at the top of TM VI likely in close spatial proximity to the previously identified interaction point, His197, at the top of the adjacent TM V. Furthermore, the data demonstrated a critical involvement in CP 96,345 binding of Lys193 and Glu194 located one alpha-helical turn above His197.