Glutamic acid decarboxylase (GAD) is a major islet cell autoantigen in insulin-dependent diabetes mellitus (IDDM), and autoantibodies are found in high frequencies in patients with recent-onset IDDM, stiff-man syndrome (SMS), and autoimmune polyendocrine syndrome type I (APS I). Antigens in autoimmune disorders are often enzymes, and autoantibody binding frequently inhibit their activity. In this study, we examined the reactivity of anti-GAD-containing sera from 7 patients with IDDM, 4 patients with SMS, and 5 patients with APS I. All sera immunoprecipitated GAD from [35S]methionine-labeled rat islet lysates and the sera from patients with SMS and APS I, but none of the IDDM patients' sera, identified the GAD protein in Western blots. Two of four SMS patients' sera and 5 of 5 APS I patients' sera, in contrast to 0 of 7 IDDM patients' sera, inhibited the enzymatic activity of GAD. When the various sera were tested with the GAD65 and GAD67 isoforms, produced separately by transient expression in COS cells, the enzymatic activity of GAD65 was inhibited by sera from patients with SMS and APS I, whereas no effect on the GAD67 activity was observed. Taken together, the results demonstrate that the GAD autoantibodies in these three disorders display marked differences in epitope recognition and indicate that, during the development of the diseases, the autoantigen is being presented to the immune system through separate pathogenetic mechanisms.