Although most individuals with insulin-dependent diabetes mellitus (IDDM) have autoantibodies to glutamic acid decarboxylase (GAD), antibodies to GAD are also present in some individuals with a low risk of developing diabetes. The GAD autoantibodies of IDDM are specific for the GAD65 isoform, do not bind denatured GAD protein, and target epitope(s) dependent on conformation of the protein. However, the IDDM epitopes have been difficult to further define because the antibodies do not bind GAD protein fragments or synthetic peptides. Since the GAD67 isoform is highly homologous to GAD65 but is usually not a target of the GAD autoantibodies in IDDM sera, we created six GAD65/GAD67 chimeric proteins to maintain the overall GAD protein conformation and used these chimeric proteins to map conformation-dependent epitopes of GAD65 targeted by IDDM sera. We find that the GAD binding present in most IDDM sera (n = 11 of 12) is composed of two distinct GAD antibody specificities that target different conformation-dependent regions of the GAD65 protein, one that is located between amino acids 240 and 435 (termed IDDM-E1) and one that is located between amino acids 451 and 570 (termed IDDM-E2). One IDDM serum (n = 1 of 12) bound only the IDDM-E1 region. Identification of epitopes targeted by IDDM sera may allow one to distinguish between GAD antibody-positive individuals at high and low risk of developing IDDM and to determine if differences in the autoimmune repertoire directed at GAD are present. The chimeric GAD65/GAD67 proteins may also be useful in designing GAD assays specific for IDDM.