We have demonstrated that there is an antibody related to extraocular muscle enlargement in autoimmune ophthalmopathy (Graves' ophthalmopathy, thyroid-associated correlated with orbital computed tomography (CT). This study was designed to identify the autoantigen and to determine whether there are common antigens among the extraocular muscle, the lacrimal gland, and the thyroid. We prepared a 100,000g sediment fraction of porcine extraocular muscle, lacrimal gland, thyroid, and human thyroid, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with sera from patients with Graves' disease, with or without ophthalmopathy, classified by symptoms and signs combined with orbital CT and normal controls. The results showed there was an approximately 55-kDa protein band which was recognized by the sera in 32.1% (9/28) of patients with autoimmune ophthalmopathy and in 47.3% (9/19) of patients with extraocular muscle enlargement demonstrated by orbital CT. It was significantly higher than the positive rates in patients without autoimmune ophthalmopathy and normal controls (15.8 and 11.1%, respectively, P < 0.025). However, there was no common antigen among the extraocular muscles, the lacrimal gland, and the thyroid. To further confirm this eye muscle-specific antigen, the approximately 55-kDa protein band was cut and solubilized from the nitrocellulose paper after SDS-PAGE, and electrophoretically transferred and used as an antigen in enzyme-linked immunosorbent assay. The absorbance was significantly higher in patients with autoimmune ophthalmopathy than patients without ophthalmopathy (P < 0.005), and normal controls (P < 0.01). Our findings suggest that an approximately 55-kDa protein may be a possible antigen in the eye muscle related to autoimmune ophthalmopathy.