We produced autoreactive T cell hybridomas that can be induced to secrete interleukin 2 by co-culture with either syngeneic splenic stimulator cells or syngeneic Ia-positive B cell-B lymphoma hybridomas. These autoreactive hybridomas arose from the fusion of pork insulin-primed lymph node T cells with the AKR thymoma BW 5147 and occurred at a higher frequency than the expected insulin-specific hybridomas. Mapping studies using recombinant strains and blocking studies using monoclonal anti-Ia antibodies localized the stimulatory determinant to the I-Ad subregion of the major histocompatibility complex. These T cell hybridomas did not appear to be directed at any foreign antigen present in the culture system because activation occurred in serum-free, insulin-free medium (Iscove's medium). Such hybridomas should prove to be a potent tool in studying the biologic significance and function of the autoreactive response.