Cortical-type thymomas are associated with myasthenia gravis (MG) in 50% of the cases. MG is caused by antibodies against the acetylcholine receptors (AChR), but additional non-AChR muscle autoantibodies such as those against titin and ryanodine receptor (RyR) are found in up to 95% of MG patients with thymoma. To elucidate the induction of non-AChR autoantibodies in thymoma-associated MG, we studied cortical-type thymomas from seven thymoma MG patients, and sera from six of them. All six had titin antibodies, and four had RyR antibodies. Titin and RyR epitopes were co-expressed along with LFA3 and B7 (BB1) costimulatory molecules on thymoma antigen-presenting cells (APC) in all thymomas. In normal thymus, the staining by anti-titin, anti-RyR, anti-LFA3, and anti-BB1 antibodies was weak and occurred exclusively in the medulla and perivascularly. Our results indicate a primary autosensitization against titin and RyR antigens inside the thymoma. In MG-associated thymoma, the mechanisms involved in the initial autosensitization against titin and RyR are probably similar to those implicated in the autosensitization against AChR. In all cases, there is an overexpression of muscle-like epitopes and costimulatory molecules indicating that the T-cell autoimmunization is actively promoted by the pathogenic microenvironment inside the thymoma.