We have studied the kinetics of interaction of N-7-(4-nitrobenzo-2-oxa-1,3,diazole)-5-aminopentanoic acid beta-(N-trimethylammonium) ethyl ester with the purified acetylcholine receptor from Electrophorus electricus. By employing a laser stopped flow fluorimeter, the full course of the kinetics was observed. The initial analysis of data yielded the following results. 1) Both association and dissociation kinetics are composed of several overlapping reaction steps. 2) Dissociation of monoliganded receptor is biphasic. This indicates the existence of two forms in equilibrium of the monoliganded receptor. 3) Displacement of fluorescent ligand by competing ligand follows a more complicated pattern. In particular, the late stages of the dissociation kinetics are increasingly slowed down the higher the concentration of competing ligand. This indicates a strictly ordered mode of association and dissociation, i.e. first on-last off for cholinergic ligands and the receptor. These and the previous finding of two agonist binding sites at the receptor (Prinz, H., and Maelicke, A. (1983) J. Biol. Chem. 258, 10263-10272) were employed to develop minimal reaction schemes that can account for the experimental data. They require two sites but four different ligand-receptor complexes and a strictly ordered mode of binding. The reaction schemes were used to simultaneously fit whole sets of association and dissociation kinetics. The fits reinforced the conclusions drawn by the initial analysis and also provided the rate constants for all reaction steps considered in the schemes. The following additional results were obtained. 4) One form of the monoliganded and diliganded receptor are formed with very large rate constants (greater than 2 X 10(8) M-1 s-1); the other complexes are formed more slowly. 5) The rapidly formed diliganded receptor (AR-A) has a rather short half-life while the other form of diliganded receptor (AR*A) develops more slowly and dominates at equilibrium. The obtained reaction schemes and rate constants invite comparison with physiological data. For example, the properties of the two forms of diliganded receptor correlate with those of the active and desensitized state of the endplate as defined by electrophysiological studies.