The neurokinin-1 receptor is characterized by sub-nanomolar affinity for substance P and 30-100 nM affinity for other substance P-related peptides, including neurokinin B and septide. We have characterized a neurokinin-1 receptor mutant containing a Y216A substitution in the fifth transmembrane segment. This receptor mutant binds substances P with sub-nanomolar affinity but loses much of its peptide discrimination capability, exhibiting 1-2 nM binding affinity for other tachykinins. Kinetic measurements of ligand binding indicate that the increased binding affinity of neurokinin B and septide for the Y216A mutant compared to the wild-type receptor is due to a 100-fold increase in the association rate constant without appreciable change in the dissociation rate constant. The substantially increased association rate constant for the Y216A mutant suggests that the mutant receptor is probably more flexible in accommodating the approaching peptide molecule. It is proposed that a major determinant of peptide specificity for the wild-type neurokinin-1 receptor is the overall conformational compatibility between the receptor and the ligand, rather than residue-specific interactions with the divergent N-terminal residues of different peptides. Furthermore, the highly conserved nature of Tyr-216 in the G protein coupled receptor family suggests that this residue may also play an important role in the receptor activation process in general.