By using an immunoprecipitation assay, we analysed reactivity of autoantibodies to human recombinant GAD65and GAD67in sera from patients with autoimmune polyendocrine syndrome Type II (APS II) with and without Type 1 (insulin-dependent) diabetes mellitus (IDDM) compared to patients with organ-specific autoimmunity. Overall antibodies to GAD65were correlated with IDDM in all study groups, whereas GAD67antibodies were associated with IDDM when APS II coexists. Antibodies to GAD65and GAD67were detected in 13 (44.8%) and 7 (24.1%) out of 29 APS II patients with IDDM, but in only 4 (13.8%) and 2 (6.9%) out of 29 APS II patients without IDDM, respectively (p<0.05). In short-standing IDDM (<1 year), antibodies to GAD67were significantly more frequent in patients with APS II (5 of 9 [55.6%] subjects) compared to matched diabetic patients without coexisting polyendocrinopathy (1 of 18 [5.6%]subjects) (p<0.02). The levels of GAD65(142±90 AU) and GAD67antibodies (178±95 AU) were significantly higher in patients with polyglandular disease than in patients with isolated IDDM (91±85 AU and 93±57 AU) (p<0.02). Interestingly, all 11 GAD67antibody positive subjects also had GAD65antibodies (p<0.0001), and in 10 of 11 anti-GAD67positive sera the GAD67antibodies could be blocked by either GAD67or GAD65, suggesting the presence of cross-reactive autoantibodies. No correlation was observed between GAD antibodies and age, sex or any particular associated autoimmune disease, besides IDDM. GAD antibodies were present in only 1 of 6 (16.7%) patients with APS Type I, in 1 of 26 (3.9%) patients with autoimmune thyroid disease but in none of the patients with Addison's disease (n = 16), pernicious anaemia (n = 7) or normal controls (n = 50). Our data suggest distinct antibody specificities reactive to GAD isoforms in APS II and IDDM, which might reflect different mechanisms of autoimmune response in IDDM with coexisting autoimmune polyendocrine autoimmunity.