We have carried out tests for antibody-dependent cell-mediated cytotoxicity (ADCC) against extra ocular muscle (EOM), Müller's muscle, orbital fibroblasts and skeletal muscle in patients with thyroid-associated ophthalmopathy (TAO) and related eye disorders. Cytotoxicity was measured as lactate dehydrogenase (LDH) release and results expressed as % cytotoxicity. Tests were positive, with EOM cells, in 65% of patients with TAO, 75% with ocular myopathy, a variant of TAO in which periorbital inflammation is minimal, 50% with euthyroid Graves' disease defined as ophthalmopathy associated with subclinical thyroiditis and in 50% of patients with stable lid lag and retraction but no other signs of progressive ophthalmopathy, but in only 13% of patients with Graves' hyperthyroidism without ophthalmopathy, 10% with Hashimoto's thyroiditis and 14% of patients with other thyroid disorders. Tests were positive, with Müller's muscle cells, in 40% of patients with TAO, 25% with ocular myopathy, 40% with euthyroid Graves' disease, 44% with lid lag, 19% with Graves'hyperthyroidism, 50% with Hashimoto's thyroiditis and in 37.5% of patients with other thyroid disorders. When skeletal muscle cells were used as target, tests were positive in 13% of patients with TAO, 31% with lid lag, 25% with Graves' hyperthyroidism and in 29% of patients with Hashimoto's thyroiditis, but in no patient with euthyroid Graves' disease or other thyroid disorders. Tests were negative in all patients and normals tested when EOM-derived fibroblasts were used as targets in ADCC. A significant positive correlation between % cytotoxicity against EOM cells and the severity of the eye muscle dysfunction expressed as an eye muscle index, was observed in patients with TAO. There was a significant negative correlation between the duration of eye disease and % cytotoxicity against EOM cells, suggesting higher titers of cytotoxic antibodies in the early stages of TAO. There was no correlation between % cytotoxicity and serum level of anti-TSH receptor antibodies, measured in a radioreceptor assay. These findings suggest that autoimmunity against Müller's muscle may play a role in the pathogenesis of persistent lid lag and retraction. The nature of the EOM and Müller's muscle autoantigens recognized by cytotoxic antibodies in the serum of patients with TAO and related eye disorders is unknown.