We have determined the prevalences of antibodies reactive with certain pig eye muscle membrane antigens, as determined from SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting, in patients with autoimmune thyroid disorders with or without associated ophthalmopathy. The most frequently detected antibody was that directed against a protein of 64 kDa. Antibodies against this antigen were detected in 33% of unselected patients with ophthalmopathy and in 75% of those with severe, active, orbital inflammation of less than 12 months duration. Such antibodies were detected also in 33% of patients with Graves' hyperthyroidism with no apparent eye disease and in 17% of patients with Hashimoto's thyroiditis with no eye disease but were not detected in patients with nonautoimmune thyroid disorders in 25 normal subjects tested. In the majority of sera showing reactivity with this protein, reactivity with membrane antigens of 55 and 95 kDa also were observed, suggesting that the three proteins were conformationally associated. Cytotoxic antibodies against human eye muscle cells, measured in antibody-dependent cell-mediated cytotoxicity (ADCC), were demonstrated in 37% of patients with ophthalmopathy. There was no significant correlation, in patients with eye disease, between positive ADCC tests and antibodies to the 64 kDa protein, the 64 kDa reactive antibody being found in 50% of patients with positive ADCC and in 35% of those with negative tests. In this study, a clear association has been shown between thyroid-associated ophthalmopathy and the detection, in immunoblotting, of antibodies reactive with a 64 kDa eye muscle membrane antigen.(ABSTRACT TRUNCATED AT 250 WORDS)