The thermodynamics and kinetics of ligand binding to the purified serotonin 5HT3 receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3, 9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluo rescien-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiological investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissociation constant of 0.32 nM. Changes in the fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissociation constants of approximately 0.2 nM. Fluorescence measurements showed that selective 5HT3 receptor ligands competed for GR-flu binding with a rank order of potency identical to that established with the radioligand [3H]-GR65630. The kinetics of GR-flu binding to the 5HT3 receptor revealed a bimolecular association process with an on-rate constant of 1.17 x 10(6) s-1 M-1 and a biphasic dissociation reaction with off-rate constants of 275 x 10(-)6 and 43 x 10(-)6 s-1. The temperature dependence of the dissociation constant yielded an enthalpic term of -26 kJ mol-1 and an entropic term of 94 J K-1 mol-1 for the binding of GR-flu to the receptor, indicating that both quantities contribute equally to the reaction. An activation enthalpy DeltaH#on and entropy DeltaS#on of binding of 50 kJ mol-1 and 43 J mol-1 K-1 were obtained, indicating that the entropy facilitates the initial steps of GR-flu binding to the 5HT3 receptor. The fluorescence anisotropy of receptor-bound GR-flu and the environmental sensitivity of the fluorescent probe suggest that the binding site has a wide entrance and that it is 0.8 pH unit more acidic than the bulk solution.