We evaluated the presence of autoantibodies to glutamic acid decarboxylase (GAD), competitive insulin autoantibodies (IAA) and islet cell antibodies (ICA) in sera from 288 non-diabetic patients with autoimmune thyroid disease (AITD), including 212 patients with Graves' disease and 76 with Hashimoto's thyroiditis, and in 235 age- and sex-matched healthy control subjects. GAD antibodies and IAA were assayed using radioimmunoassay with 125I-labelled purified pig brain GAD and human insulin, respectively. Titers of greater than 4.7 units for GAD antibodies and 50 nU/ml for IAA, respectively, the mean + 3SD of 235 age- and sex-matched healthy individuals, were defined as positive. The mean titers of GAD antibodies in patients with Graves' disease and in patients with Hashimoto's thyroiditis were 3.6 +/- 4.6 (mean +/- SD, range 0.6-52.0) units and 3.2 +/- 1.4 (range 0.6-10.0) units, respectively. Titer of GAD antibodies in patients with AITD was significantly higher than in healthy controls (P < 0.0005). Thirteen of 212 (6.1%) patients with Graves' disease and 6 of 76 (7.9%) patients with Hashimoto's thyroiditis had positive GAD antibody titers, whereas titers in healthy control sera were < 4.7 units in all but two individuals (P < 0.005). In competition analysis with purified unlabelled GAD, binding tracer was inhibited in all of 13 GAD antibody-positive Graves' sera and 5 of 6 GAD antibody-positive sera from patients with Hashimoto's thyroiditis. Eight of 212 (3.8%) patients with Graves' disease and 3 of 76 (3.9%) patients with Hashimoto's thyroiditis, but none of healthy controls had IAA levels exceeding the range for normal controls (P < 0.005). Positive IAA levels ranged between 50 and 2383 nU/ml. Strikingly, all of 19 GAD antibody-positive sera were negative for IAA. ICA were not detected in any of the patients or healthy controls. These data demonstrate that GAD antibodies in sera of AITD patients are of low titer but significantly elevated compared to healthy controls, and are independent of the appearance of IAA. They also indicate that, in patients with AITD, an autoimmune response to GAD may occur with no relationship to production of IAA.