Patients with myasthenia gravis (MG) and thymoma have in their sera antibodies which react with non-receptor antigens from striated muscle. The purpose of this investigation was to characterize the antigen(s). Polypeptides in homogenates from rat skeletal muscle were separated by SDS-PAGE and trans-blotted to nitrocellulose. Sera from six patients with MG and thymoma stained a large (molecular weight greater than 500 kD) polypeptide, while no staining was observed with sera from 20 non-thymoma MG patients. Titin is one of the large (greater than 500 kD) polypeptides of striated muscle and the antibody containing MG sera have antibodies that bind to titin in a preparation of myofibrillary proteins from rabbit skeletal muscle. The staining pattern is identical to that obtained with antiserum to titin, showing that the antigen has the same electrophoretic mobility as titin. Antibodies from the sera of the patients with MG and thymoma, affinity-purified on the large polypeptide, reacted with skeletal muscle sections in a cross-striational pattern, near the A/I band junction but within the I band, corresponding to the localization of one of the epitopes of titin. Our findings therefore indicate that the muscle antibodies found in the sera from some MG patients with thymoma are directed against titin.