The murine syngeneic mixed lymphocyte reaction (sMLR) is a T cell proliferative response to self-antigens on non-T stimulator cells. The nature and specificity of the stimulating antigens was investigated using secondary MLR. Lymph node cells were stimulated in vitro by syngeneic spleen cells. After optimal proliferation occurred, the responding cell population was recovered and restimulated with spleen cells from mice of various haplotypes. The secondary response was found to be specific for the haplotype of the primary stimulator (a self-restricted response). The use of recombinant mice in the secondary stimulation localized the determinant to the I-A and/or I-E subregions of the major histocompatibility complex (MHC). This result was confirmed by the ability of monoclonal antibodies directed to the I-A or I-E subregion products to significantly inhibit the response while alloantiserum directed to products of the K or D regions had no effect.