The acetylcholine receptor from Torpedo californica was labeled by reaction with the fluorescent probe 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole without apparent effect on its in vitro ligand binding and functional properties. Addition of acetylcholine or carbamoylcholine to the labeled-receptor preparations enhanced the fluorescence of the bound probe, and this effect was specific for agonists and inhibited by prior incubation with excess alpha-bungarotoxin. Equilibrium fluorescence titrations gave apparent dissociation constants of 0.86 +/- 0.14 mM for carbamoylcholine and 79 +/- 11 microM for acetylcholine, in good agreement with the dissociation constants measured for the permeability response of the receptor. Stopped-flow experiments showed that the fluorescence change was a single exponential process whose rate increased with ligand concentration, reaching a saturating value for carbamoylcholine of approximately 400 s-1. The equilibrium binding of carbamoylcholine was not significantly affected by prior incubation of the receptor with d-tubocurarine or histrionicotoxin and the dissociation constant was only slightly increased in the presence of lidocaine. These inhibitory ligands do not, therefore, compete directly with agonists for this low-affinity binding site, suggesting that their mode of action may be indirect.