The eye changes associated with Graves' hyperthyroidism can be classified into two subtypes, congestive ophthalmopathy (CO), in which inflammatory changes in the periorbital tissues predominate, and ocular myopathy (OM), in which eye muscle damage is the main feature. Antibodies against the flavoprotein (Fp) subunit of succinate dehydrogenase (SDH), the 64-kd protein, and G2s, a thyroid and eye muscle shared protein of unknown function, are good markers of eye muscle cell damage in patients with OM. Another antigen associated with ophthalmopathy is the flavine adenine nucleotide (FAD) cofactor of several mitochondrial enzymes, including SDH. We tested for serum antibodies against purified human recombinant Fp, FAD, and a G2s fusion protein, in patients with thyroid-associated ophthalmopathy (TAO) and control patients and subjects, in enzyme-linked immunosorbent assay. Antibodies against Fp were detected in 32% of patients with TAO, 30% with Graves' hyperthyroidism (GH), 16% with Hashimoto's thyroiditis (HT), in 14% of patients with multi-nodular goiter (MNG), and in 6% of normal subjects. Antibodies against FAD were found in 24%, 30%, 24%, and 14%, respectively, of these patients and in 12% of the normals, while antibodies against G2s were detected in 50% of patients with TAO, 40% with GH, 40% with HT, in 29% of patients with MNG, and in 7% of normals. We also tested for antibodies against SDH, FAD, and G2s in 12 patients with GH who developed CO (6 patients) or OM (6 patients) after treatment with antithyroid drugs. Of the 6 patients who developed OM, antibodies against SDH preceded the onset of eye disease in 4 and coincided with it in 2, antibodies against G2s preceded eye muscle disease in 5 and coincided with it in 1 patient while antibodies against FAD preceded the development of OM in 5 patients. Of the 6 patients who developed CO, antibodies against SDH were detected in only one patient and borderline levels were demonstrated in 1, while anti-FAD and anti-G2s each preceded the onset of eye signs in 6 patients. Positive sera from another group of patients with TAO, and a second group of normal subjects, were tested at increasing serum dilutions. Sera from the two groups showed similar dilution patterns, except for a few patients with TAO in whom increasing dilutions was associated with increased, then decreased, antibody levels. In this experiment the prevalences of the two antibodies were much greater in patients with TAO namely, 67% for anti-Fp and 89% for anti-G2s, while the prevalences in the normals were 11% and 22%, respectively. The reason for this apparent discrepancy is not clear but may reflect subject and assay differences. Because Fp is found within the mitochondrial membrane it is likely that the corresponding antibodies are produced after eye muscle necrosis, and do not play a role in its pathogenesis. The primary reaction in the eye muscle may be T-cell autoimmunity against G2s, although this has not been proven. The mechanism for the production of antibodies against G2s, FAD, and Fp in subj...