The binding properties of a new radioligand, [methyl-3H]-(-)-(R)-5-[(1-methyl-1H-indol-3-yl)carbonyl]-4,5,6,7- tetrahydro-1H-benzimidazole monohydrochloride ([3H]YM060), were studied in membranes of the rat cerebral cortex. [3H]YM060 rapidly associated with its binding sites in membranes and reversibly dissociated. Saturation analysis revealed that the specific binding of [3H]YM060 was saturable and non-specific binding was low. Scatchard analysis yielded a linear plot, suggesting a single population of binding sites with a dissociation constant (Kd) of 8.4 +/- 0.2 pM (n = 3) and the kinetic Kd determined from the association constant (K+1) and the dissociation rate constant (K-1) was similar. The maximum number of binding sites (Bmax) was 37.0 +/- 0.8 fmol/mg protein (n = 3). [3H]YM060 binding was potently and stereospecifically inhibited by serotonin (5-HT)3 receptor agonists and antagonists. Other 5-HT receptor ligands such as 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), methysergide and ketanserin were inactive to inhibit specific binding at 10(-4) M. These results suggest that [3H]YM060 is a highly potent and selective 5-HT3 receptor radioligand and will be useful in the further analysis of 5-HT3 receptors.