The human muscle acetylcholine receptor (AChR) is an oligomeric membrane protein consisting of (alpha1)2,beta,delta,epsilon subunits in the adult form and (alpha 1)2,beta,gamma,delta in the fetal form. The adult AChR is the target for autoantibodies in myasthenia gravis (MG), and antibodies that block the function of fetal AChR can cross the placenta and paralyse the developing baby causing joint contractures. Monoclonal antibodies (mAbs) raised against purified AChR were characterised previously in terms of binding to five regions, three of which appeared to partially overlap, but the subunit localisation of the regions was not clearly established and they were assumed to be mainly on the immunodominant alpha subunits. We have studied binding of the mAbs to AChR subunit extracellular fragments expressed in E. coli, and to AChRs derived from TE671 cells and from fibroblast cell lines expressing human/Torpedo and Torpedo/mouse hybrid receptors. Using a combination of Western blotting and immunoprecipitation experiments, we demonstrate the subunit specificity of each mAb. The results confirm our previous observations but importantly show that only two of the regions are on the alpha subunit, the three others being on the beta, gamma and delta subunits of human AChR. Thus these mAbs should be useful in studies of AChR subunit expression in normal and diseased tissue, and to define further the binding sites of antibodies in MG patients.