The a-subunit of the nicotinic acetylcholine receptor is the major target of the autoimmune response in myasthenia gravis. We investigated the proliferative response of T cells from patients with myasthenia gravis and healthy volunteers to recombinant polypeptides of the human acetylcholine receptor including the full-length a-subunit (al-437). T cells from 20 (71%) of 28 patients and 7 (37%) of 19 healthy volunteers responded in primary cultures. Subsequently, specific T-cell lines were established: CD4+, C D F , UCHLl+, and major histocompatibility complex (MHC) class 11-restricted. Using a set of fragments of the a-subunit, major antigenic sites could be localized on the extracellular, N-terminal part of the molecule as well as close to the C-terminus. The T-cell response was heterogeneous, both among different individuals and among T-cell lines from a single donor. These T cells did not cross-react with Torpedo acetylcholine receptor, which was previously used as a substitute for human muscle acetylcholine receptor, suggesting that the T cells had a bias for unique human sequences. A single antigenic fragment could be presented in the context of different MHC class 11 molecules, and different fragments could be presented in the context of the same MHC molecule. This supports earlier observations of considerable heterogeneity in dealing with acetylcholine receptor as an autoantigen on the level of both T cells and antigen-presenting cells. The data also demonstrate that acetylcholine receptor-specific T cells are present in the normal immune repertoire, and emphasize the role of immune regulation for maintaining a state of tolerance.