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1: This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. 2: Specific binding of [3H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with Kd values of 3.76±0.45 and 5.03±0.31?nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0?pmol?mg protein-1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. 3: Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin-A displaying a ~five-fold higher affinity than orexin-B (Ki values of 318±158 and 1516±597?nM, respectively). 4: SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5?nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2?nM, respectively) formats. 5: Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. 6: These studies indicate that [3H]SB-674042 is a specific, high-affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors.British Journal of Pharmacology (2004) 141, 340-346. doi:10.1038/sj.bjp.0705610 [ABSTRACT FROM AUTHOR]