Mutational analysis of the NK-1 receptor indicates that residues involved in non-peptide antagonist binding cluster around the outer portion of transmembrane segments (TM) V and VI. In contrast mutations affecting the binding of the natural peptide agonist, substance P, are scattered in the exterior part of the receptor. Recently it was reported that a number of mutations in TM-II also seriously impair substance P binding. Here we confirm that Ala substitutions for these residues located on a hydrophilic helical face of TM-II basically eliminate substance P binding to the NK-1 receptor, provided that a radiolabeled non-peptide antagonist is used as radioligand. Surprisingly, radiolabeled substance P bound well to all these mutant receptors and was displaced with only slightly reduced affinity by the unlabeled peptide and by the non-peptide antagonists. The wild-type homologous NK-2 receptor displayed properties similar to those observed in the mutated NK-1 receptors, i.e. concomitant high affinity binding of radiolabeled agonist peptide (in this case neurokinin A), yet low affinity, G-protein independent competition of unlabeled peptide with radiolabeled non-peptide antagonist. It is concluded that substitutions in TM-II of the NK-1 receptor do not affect the high affinity binding of substance P but instead block the ability of the peptides to compete for non-peptide antagonist binding. It is suggested that certain mutations can impair interchange between receptor conformations that each bind different ligands with high affinity.