Type 1 diabetes is a major histocompatibility complex (MHC) class II-associated autoimmune disease mediated by beta-cell-specific T-cells and characterized by circulating autoantibodies to beta-cell molecules. In the BB/Wor diabetes-prone (DP) rat, type 1 diabetes develops spontaneously with an incidence of >90%. BB diabetes can be adoptively transferred to naive syngeneic or MHC class II-compatible rats with islet cell-activated T-cell lines derived from diabetic BB/Wor rats. However, the target beta-cell autoantigen(s) in BB diabetes has not yet been defined. BB rat T-cell lines activated in vitro with antigen-presenting cells (APC) and BB islet cell crude membranes (CM), but not islet cell cytosol, adoptively transfer diabetes into young DP recipients. To determine if the target autoantigen is an integral or peripheral membrane protein, islet cell CM were treated with 0.5 mol/l KCl or 0.2 mol/l Na2CO3 (pH 11). Both treatments selectively extract peripheral proteins from the cell membrane without affecting the disposition of integral (transmembrane) proteins. T-cell lines activated in vitro with APC and 0.5 mol/l KCl, or pH 11 (0.2 mol/l Na2CO3)-treated islet cell CM, transferred diabetes into young DP rats. Conversely, T-cell lines activated in vitro with APC and the supernatant of 0.5 mol/l KCl-treated CM (containing extracted peripheral proteins), did not adoptively transfer diabetes. After activation in vitro with islet cell membrane antigens, the diabetes-inducing cell lines were comprised of both CD4+ CD8- T-cells and 10-30% B-cells. We conclude that a major CD4+ T-cell target autoantigen in BB diabetes is a membrane-associated beta-cell molecule with the characteristics of an integral beta-cell membrane protein. The identification of this MHC class II-restricted beta-cell target molecule will allow the design of antigen-specific intervention protocols to prevent the onset of type 1 diabetes in genetically susceptible individuals.