An enzyme-linked immunoabsorbent assay has been developed for measuring anti-acetylcholine receptor antibodies from sera of patients with myasthenia gravis or tissue culture supernatants from hybridomas. Acetylcholine receptor from a detergent extract of muscle tissue was bound indirectly to microtiter plates via a monoclonal anti-receptor antibody already coupled to the polyvinyl plates. Myasthenic sera or antibodies in tissue culture media were then tested for binding to the acetylcholine receptor attached to the monoclonal antibody. Anti-receptor antibodies were detected in the serum of 80% of myasthenic patients when assayed by this method and the levels of antibody corresponded fairly well with antibody titers determined by an immunoprecipitation assay. Occasional myasthenic patients had serum antibodies which reacted specifically with the monoclonal antibody attached to the microtiter plate. The assay described here was far less time-consuming than immunoprecipitation assays, required only small quantities of receptor, and did not require the use of radioisotopes such as 125I-α-bungarotoxin. © 1983.