With the reversible sequential (ReSeq) binding assay, we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast assocn.-dissocn. kinetics. We chose the nicotinic-acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equil., kinetic, and competition-binding assays using fluorescent derivs. of the antagonist a-conotoxin GI (a-CnTx). Thereby, we detd. the binding consts. of unlabeled a-CnTx and d-tubocurarine. The high selectivity of a-CnTx for muscle-type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand-binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq-binding assays even at low receptor-expression levels. We expect our approach to be of generic importance for functional screening of compds. or membrane receptors, and for the detailed characterization of rare primary cells. [on SciFinder (R)]