The use of in vitro assays of cell-mediated immunity has provided evidence of a defect in thyroid antigen-specific T suppressor cells in the peripheral blood of patients with autoimmune diseases. We employed a direct assay of T lymphocyte migration inhibition in an attempt to demonstrate cell-mediated immune responses to thyroid antigens in untreated patients with hyperthyroid Graves' disease and examined the influence of (1) a variety of antigen preparations, (2) different assay conditions, and (3) the HLA-DR3 status of normal subjects on this system. There was no significant difference in the migration of lymphocytes from 13 untreated patients with hyperthyroid Graves' disease to a crude 800 X g supernatant antigen preparation of normal human thyroid when incubated at either 20 or 37 C compared with the response of 13 normal subjects. The mean migration index was 57 +/- 22 (+/- SD) in the Graves' patients compared with 65.2 +/- 19.9 in the normal subjects at 20 C and 83 +/- 16.9 in the Graves' patients compared with 86.6 +/- 15.1 in the normal subjects at 37 C. Similarly, there was no significant difference in the migration indices obtained with T cells from Graves' patients and normal subjects using an 800 X g supernatant prepared from the thyroid glands of 2 patients with Graves' disease, incubated at 20 or 37 C (44.0 +/- 22.5 in the Graves' patients compared with 45.7 +/- 12.8 in the normal subjects at 20 C and 67.9 +/- 20.8 in the patients compared with 72 +/- 12.6 in the controls at 37 C). In contrast, the migration indices calculated for 20 C incubation were significantly lower than the corresponding value at 37 C using 800 X g normal thyroid antigen (P less than 0.05) and 800 X g Graves' thyroid antigen (P less than 0.01) in both patient and control groups. An identically prepared uterine antigen produced a similar reduction in the migration indices at 20 C compared with those at 37 C in 6 normal donors (P less than 0.05), but no temperature effect was found when 8 normal subjects were tested with purified protein derivative of tuberculin. The responses of 12 normal individuals to 800 X g supernatants of the normal and Graves' thyroid antigens were not influenced by the HLA-DR3 antigen.(ABSTRACT TRUNCATED AT 400 WORDS)