Antibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction are detectable in most patients with myasthenia gravis (MG) and assumed to participate in the destruction of the AChR, thereby, causing the characteristics signs and symptoms of the disease. The extent and importance of T cell responses to AChR and its subunits in MG are still unsettled. We have now examined T cell reactivities using human recombinant AChR-alpha subunit as antigen. Upon recognition of appropriate antigen in an MHC-class II-restricted fashion, memory T cells secrete interferon-gamma (IFN-gamma). Adopting this principle in an immunospot assay we found that 73% of MG patients had recombinant human AChR-alpha subunit-reactive T cells at a median value of 1 per 56,000 blood mononuclear cells, while only 27% of the MG patients responded to the alpha subunit in a conventional lymphocyte proliferation assay. This compares with even lower numbers of AChR-reactive T cells and 14% positivity in the proliferation assay among control subjects. The T cell responses to the control antigens purified protein derivative and myelin basic protein did not differ between MG and controls, underlining the specificity of an augmented T cell reactivity to AChR-alpha subunit in MG. Alpha Subunit-specific T cell lines and clones propagated from patients with MG and healthy controls yielded a high proportion of alpha subunit-reactive T cells in the IFN-gamma immunospot assay. Their appearance was inhibited by the addition of monoclonal anti-MHC class II antibodies, demonstrating that an MHC-restricted T cell response was measured. Our data underline that the AChR-alpha subunit is a major T cell autoantigen in MG.