A fluorescently labeled ligand for the 5-HT3 serotonin receptor was synthesized and its sub-nanomolar affinity for the purified, detergent solubilized receptor was measured. The change in the ligand's fluorescence upon receptor binding was used to directly measure its dissocn. const. for receptor binding, to det. the pharmacol. of the receptor, and finally to characterize the binding site of the receptor. A total internal reflection fluorescence (TIRF) assay for the 5-HT3 receptor was developed, which is suitable for high-through-put screening. Therefore, the receptor was immobilized via its C-terminal His-tag onto a nitrilotriacetic acid-modified quartz surface. The affinities of both the fluorescent ligand and several non-fluorescent compds. were rapidly detd. by the TIRF assay, and were shown to agree well with both the soln. and classical radioligand binding assays. This indicated that the functional integrity of the receptor was preserved at the sensor surface. Due to the extreme sensitivity, the TIRF assay allows a complete pharmacol. affinity profile of a quantity of receptor provided by a small no. of highly-expressing cells. [on SciFinder (R)]