Autoantibodies to glutamic acid decarboxylase (GAD) are useful diagnostic and predictive markers for Type 1 (insulin-dependent) diabetes mellitus. In the present study we describe a development of simple, reproducible, and quantitative radioimmunoassays for detecting GAD65 and GAD67 antibodies, and compare sensitivity and specificity of these assays with native GADAb radioimmunoassay. We used in vitro transcribed and translated recombinant human islet GAD65 and GAD67 as antigens, and anti-human IgG was used to separate free from antibody-bound ligand. By using these assays, GAD65Ab and GAD67Ab were detected in 65% and 25% of recent-onset Japanese patients with Type 1 diabetes, respectively, but none of 71 healthy control subjects tested were postive for GAD65Ab and GAD67Ab. Moreover, none of 48 patients with other autoimmune disease had GAD65Ab or GAD67Ab. There was a 100% correlation between the sensitivity and specificity of GAD65Ab assay and native GADAb assay. GAD65Ab and GAD67Ab were concordant in 28% of Type 1 diabetic sera and the levels of GAD65Ab in doubly positive patients were significantly higher than those in only GAD65Ab positive patients (P < 0.01). GAD65Ab are specific markers for Type 1 diabetes, and the radioimmunoassay using in vitro translated GAD and anti-human IgG, which is sensitive, convenient and low cost for detecting GAD antibodies, will facilitate large population screening of Type 1 diabetes.