The aim of this work was to characterise the eye muscle antigens reacting with autoantibodies from Graves ophthalmopathy patients to elucidate the function of these antibodies. As estimated by ELISA test antibodies of IgG class reacting with porcine microsomal membranes are present in about 25% of patients while of IgM class in about 15% of patients with Graves ophthalmopathy. Their presence do not correlate with ophthalmopathy index, neither they have relation to treatment. Anti eye muscle antibodies were present at some stages of the disease in three patients who develop ophthalmopathy, from the group of 26 patients treated during one year for hyperthyroidism. However, sporadically these antibodies were found also in about 20% of patients with Hashimoto disease, Lupus erythematosus or Scleroderma. Some of them cross react with antigens of skeletal muscle and liver. Eye muscle antigens reacting with patients antibodies are localised in plasma membranes and in membranes of smooth reticulum. Affinity purification of solubilised porcine eye muscle membrane proteins on a column with immunoglobulins from pooled serum of patients resulted in 23 fold purification of the antigen. The sensitivity of ELISA was not significantly increased by the use of affinity purified antigen, however some of previously negative ophthalmopathy sera gave positive reaction. Porcine eye muscle membrane proteins were separated by SDS PAGE and transferred to nitrocellulose. The reactions of electroblotted proteins with sera from patients with Graves ophthalmopathy and also sera from healthy controls shown very complex pattern. There was not a single antigen or antigens reacting only with antibodies present in sera of ophthalmopathy patients and not in controls. Patients sera reacted more often than control sera with an antigen of about 40 kDa. The reaction of sera from some patients with proteins about 100, 70 and 65 kDa were stronger than between these proteins and control sera. No changes in the pattern of reaction between antibodies and eye muscle antigens were noticed in serum of the same Graves' patient with or without ophthalmopathy during one year follow up and treatment, regardless of clinical course of the disease. When human eye muscle membrane fractions from tissue obtained during strabismus repair or at autopsy was used for immunoblotting, smaller number of proteins reacted with autoantibodies. Again there was no single antigen or antigens reacting with antibodies from sera of all Graves ophthalmopathy patients. Sera of some patients reacted with antigens about 50 kDa, not recognized by controls. The results of present study show, that the anti eye muscle antibodies are present in some of Graves ophthalmopathy patients.(ABSTRACT TRUNCATED AT 400 WORDS)