The equilibrium and kinetic properties of agonist binding to the membrane-bound acetylcholine receptor from Torpedo californica have been measured by the fluorescence changes of a probe, 4-[[(iodoacetoxy)ethyl]methylamino]-7-nitro-2,1,3-benzoxadiazole, which was covalently bound to the receptor protein. Dissociation constants for the binding of several agonists have been measured in fluorescence titration experiments, and these are in good agreement with apparent equilibrium constants obtained from the concentration dependence of the cation flux response measured in quantitative in vitro kinetic experiments. These results provide evidence for the existence of a low-affinity binding site for agonists which is likely to be a functionally important site for channel activation. The kinetics of carbamylcholine and acetylcholine binding to this site have been measured in stopped-flow fluorescence experiments. Kinetic traces were recorded over a wide range of agonist concentrations, and all could be fit by a single exponential process whose rate and amplitude increased hyperbolically with the concentration of ligand. The observed signal change has been ascribed to a conformational transition of the receptor-ligand complex, and this occurs on a millisecond time scale at saturating ligand concentrations which is sufficiently fast to suggest a role for this binding site in the process of channel activation. These results indicate that in the Torpedo AcChR activation and desensitization may be parallel processes which are mediated by agonist association with different receptor binding sites.