We investigated whether a novel immunisation scheme using an endogenous protein could stimulate an autoreactive cytolytic response. The protein selected was porcine dihydrolipoamide dehydrogenase, the E3 component of the mitochondrial 2-OADC enzyme family, because it is structurally conserved in mammals and ubiquitously expressed. Recombinant insulin was used as an alternative antigen. Female BALB/c mice were injected with adjuvant-free syngeneic lymphoid cells that had been exposed to E3 in hypertonic medium to facilitate its pinocytosis and were given two booster injections. Effector lymphoid cells from immunised mice were cultured in vitro with irradiated syngeneic cells that had been treated with hypertonic medium, either with or without antigen. Cytolytic effector cells were detected that lysed isogeneic and not allogeneic target cells, but only from mice immunised with E3. This experimental system provides a new model for the early stages of the development of autoimmunity.