To investigate the presence of antibodies in myasthenia gravis (MG) patients, we have developed a new reproducible and sensitive enzyme immunoassay (EIA-AChR), in which a beta subunit-specific monoclonal antibody (mAb 73) immobilizes fetal calf acetylcholine receptors (AChRs). We tested 92 MG patients (42 with positive and 50 with negative antibody titers), 60 healthy controls, and 40 controls with other autoimmune diseases. EIA-AChR detected immunoglobulin G (IgG) titers in all of the seropositive samples, with a significant correlation between these and those obtained using the traditional immunoprecipitation method. Moreover, 5 seronegative patients at immunoprecipitation assay were positive at EIA-AChR. EIA-AChR was also useful in revealing: (1) a seropositive patient subpopulation with generalized MG who had Abs directed against alpha-Bungarotoxin binding sites; and (2) patients with IgM directed against fetal calf AChR (detected in 13 seronegative and 16 seropositive MG patients, and in 6 of the patients with other autoimmune diseases).