An agonist such as carbamylcholine or phenyltrimethylammonium induced a second, slower complete inactivation of acetylcholine receptor prepared from Electrophorus electricus. The rate of this inactivation of the receptor followed first-order kinetics. The rate constant of the inactivation increased with the agonist concentration until it reached a plateau, the value of which was 0.19 h-1 at 4.5 degrees C. The reaction was also temperature dependent, and the activation energy of the inactivation caused by 1 mM carbamylcholine was estimated to be 7.6 kcal/mol. The inactive receptor was reconverted to the active form with a rate constant of about 0.015 h-1 at 4.5 degrees C when the carbamylcholine concentration (0.1 mM) was reduced by 15-fold dilution in eel Ringer's solution. These results can be interpreted by adding, to the minimal reaction scheme proposed by the Hess group, a second, slower, reversible inactivation process either through the intact form or through the first desensitized form of the receptor binding two agonist molecules.