CYTOTOXIC T lymphocytes generated during a unidirectional mixed lymphocyte culture (MLC) lyse target cells which have the antigenic phenotype of the allogeneic stimulating cells. The cytotoxic effect is restricted to cells that bear the same major histocompatibility antigens (H–2 antigens in mice) as the stimulating cells1. H–2 restriction of cytotoxicity is also seen when immune T lymphocytes react in vitro to cells bearing viral2, chemical3, or minor histocompatibility antigens4 or following autosensitisation against unmodified fibroblasts5. Thus, H–2 restriction of cytotoxic lymphocytes has been observed in many circumstances. We now report that cultures of normal spleen cells generate T lymphocytes that damage target cells regardless of their H–2 phenotype.