Antibodies to islet antigens are useful markers of the pathological process that results in destruction of beta cells in type I diabetes. The targets of these antibodies, however, are not well characterized and their role in the pathological process remains to be established. To better understand the range of antigens recognized by autoantibodies in type I diabetes mellitus, we carried out immunoblotting on protein extracts from human islets and rat insulinomas. Using this approach high titer antibodies specific for islet antigens were detected in 20/28 sera from recent onset type I diabetics and were infrequent (4/28) in sera from age-matched controls. Sera from diabetics reacted with multiple antigens at titers from 1/100–1/4000 while control sera usually bound a single band at lower dilutions. Although antigens of M, 52, 84, 116 and 150 kilodalton were recognized most frequently, no antigen was bound by more than 50% of diabetic sera. Some of these antigens enriched in the membrane fraction while others were not. The data demonstrate that a heterogeneous group of islet antigens is recognized by autoantibodies present at the onset of type I diabetes when islet destruction is complete. These findings contrast previous reports using immunoprecipi-tation with undiluted sera that principally identify a 64 kDa islet antigen. Thus, the immunoblot technique detects a different set of reactivities than previously identified by immunoprecipitation. The pattern of multiple reactivities with both surface and cytoplasmic antigens suggest that many autoantibodies may be generated by beta cell destruction and some of these may amplify the ongoing immune response.