We have shown that SR 48968 possess sub-micromolar affinity for the human tachyknin NK3 receptor; however, its affinity for the rat NK3 receptor is greater than 10 microM. To determine the functional domain(s) responsible for the species variation in binding affinities, we have constructed several human/rat chimeric NK3 receptors. Based on studies of these chimeric receptors, the species-specific binding sites for SR 48968 were localized to five residues in the 1st and 2nd transmembrane segments of the human NK3 receptor. We have individually mutated all five residues in the rat receptor to their corresponding residues in human. Only two single-substituted mutants (V121M and G133A) show a small increase in their binding affinities for SR 48968. However, a mutant containing both substitutions was shown to have the same affinity for SR 48968 as the wild type human NK3 receptor. It is concluded that collectively these two amino acid changes are responsible for the species difference in binding affinities for SR 48968.