The mouse monoclonal antibody (m.ab) binding sites on human acetylcholine receptor have been mapped by inhibition by F(ab')2 m.ab fragments, by competition with a rat m.ab against the main immunogenic region (anti-m.i.r), and by their ability to protect the a-Bungarotoxin (a-BuTx) binding sites from inhibition by a myasthenia gravis (MG) plasma. Two m.abs (C3 and D6) that bind to two distinct but overlapping regions on the AChR, were inhibited by the anti-m.i.r. m.ab M35. M.abs binding to three other regions protected the a-BuTx sites by up to 50%. In further inhibition assays these m.abs were used to define the binding sites for MG anti-AChR antibodies. There was considerable heterogeneity in the antigenic specificity of the MG sera. The inhibition of MG anti-AChR by anti-m.i.r. M35 correlated highly with inhibition by mouse m.ab D6, but not with inhibition by m.ab C3. There was a correlation between inhibition by m.abs F8 and B3, although these m.abs bind to two non-overlapping regions. Some MG anti-AChR antibodies bound to epitopes that only partially overlapped those defined by m.abs. Inhibition of human antibody binding by m.abs raised against specific antigens is a useful approach that should help define the epitopes to which autoantibodies bind.