Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the N ␣ position were synthesized and characterized. Peptides modified at the ␣-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18,000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyllabeled peptides indicating that the micro-environment of the binding site is more hydrophobic for the nonpeptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl-or fluoresceinyllabeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.Many peptide hormones and neuropeptides act via known receptors belonging to the superfamily of G protein-coupled receptors characterized by a seven membrane-spanning topology. There is considerable interest in understanding ligandreceptor recognition and the mechanisms of action of both non-peptide ligands and natural peptides for peptide receptors. The tachykinin substance P (SP) 1 is a peptide transmitter that plays an important role in pain perception and neurogenic inflammation (1, 2). The cellular actions of SP are mediated by the tachykinin (neurokinin) NK1 receptor, a G protein-coupled receptor. Therefore, the NK1 receptor has been the target for the development of multiple non-peptide antagonists. The prototype NK1 non-peptide antagonist is the quinuclidine compound CP96,345, which acts as a high affinity and highly selective non-peptide inhibitor of SP in both binding and functional a...