Acetylcholine receptor-phosphorylation has been compared in sealed and lysed right-side-out membrane vesicles prepared form Torpedo californica electric organ. Phosphorylation was increased 5- to 12-fold in hypotonically lysed vesicles as compared with untreated vesicles. Control experiments confirm that this enhancement is a result of increased permeability of the membrane to ATP. These data suggest that the acetylcholine receptor kinase is located on the cytoplasmic side of the plasma membrane. Results with detergent lysis support this conclusion. Although the acetylcholine receptor constitutes less than 10% of the total protein in these membranes, the kinase was found to be highly specific for polypeptides corresponding in molecular weight to acetylcholine receptor subunits.