The Salk Institute for Biological Studies, San Diego, California 92138, and Department of Molecular Biology, Abbott Laboratories, Abbott Park, North Chicago, Illinois 60064 Received November 4, 1985; Revised Manuscript Received January 16, 1986 abstract: The sequence a 127-143 of the a subunit of the acetylcholine receptor has been proposed to contain several important features:(1) the acetylcholine binding site,(2) the onlyN-glycosylation site of the a subunit, at asparagine-«141, and (3) two cysteine residues, at al28 and al42, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide «127-143 cyclized by a disulfide bond between a 128 and a 142. Antisera to receptor and its a subunit were able toimmunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor, a-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with A-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher than the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence a 127-143 (1) is not fully exposed on the surface when the receptor is in the membrane,(2) is probably glycosylated at asparagine-«141,(3) may have cysteines-«128 and-«142 forming a disulfide bond which is notrelated to the disulfide known to be close tothe cholinergic binding site, and (4) is not involved in the formation of the binding site for cholinergic agonists or antagonists.