Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectively a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be mediated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-I) surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.