Recent studies in NOD mice suggest that cellular and humoral responses against beta cell protein(s) cross-reactive with mycobacterial heat-shock protein, hsp60, are implicated in the development of autoimmune diabetes. However, this putative, hsp60-related autoantigen has not yet been identified nor have the preceding events triggering the autoimmunity against it. Our recent studies show that antibodies to the mammalian hsp60 bind specifically to the 62 kDa protein located to insulin secretory granules and mitochondria of pancreatic beta cells of healthy mice . In islets of prediabetic NOD mice affected by insulitis, the cellular distribution of this hsp60-related antigen was found to be altered. In the present report, we have examined whether this endogenous hsp60-related protein of secretory granules serves as an autoantigen in type I diabetes. The results of Western blot analysis indicate that diabetic mice sera show reactivity to a 62 kDa islet cell antigen. The NOD mice sera that were positive in detection of the 62 kDa islet cell antigen were also able to recognize the recombinant human hsp60. Immunogold electron microscopy revealed that diabetic NOD mouse sera, cross-reactive to human recombinant hsp60, recognize the antigen located in secretory granules of beta cells. Double-immunogold labelling demonstrated that antigens recognized by both diabetic NOD mice sera and monoclonal hsp60 antibodies co-localized in the same secretory granules of beta cells. Preincubation of islet cell sections with one type of antibody blocks subsequent binding of the other, indicating that epitopes recognized by both antisera on these proteins are shared. Moreover, preadsorption of diabetic sera with the recombinant human hsp60 abolished labelling of secretory granules. These results indicate that the hsp60-related protein of beta cell secretory granules is an autoantigen in type I diabetes in NOD mice.