Glutamic acid decarboxylase autoantibodies may aid in rapid screening strategies predicting IDDM before clinical onset. Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets. Because human IDDM involves beta-cell-specific autoimmunity, we tested 190 new IDDM patients and 51 healthy control subjects for antibodies to recombinant human islet GAD65, rat islet GAD67, or human insulinoma/cerebellum GAD67, each expressed separately in hamster fibroblasts. By using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometric fluorogram scanning, 132 of 190 (70%) of new IDDM patients had GAD65 autoantibodies, whereas only 17 of 190 (9%) had antibodies to rat GAD67 (P < 0.001). Of healthy control subjects, 2 of 51 (3.9%) and 1 of 51 (1.9%) had antibodies to GAD65 and GAD67, respectively. All 17 GAD67 antibody-positive patients also had GAD65 antibodies; 14 of 17 with greater GAD65 than GAD67 index. Control studies showed comparable reactivity between recombinant rat and human GAD67 and between different subcellular preparations of recombinant GAD67 of either species. In conclusion, only GAD65 is expressed in human islets, the autoantibody response is primarily to this isoform, and GAD67 antibodies add little to IDDM detection.