P atients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.