In vitro sensitized cytotoxic T lymphocytes (CTLs) primarily detect antigens controlled within the major histocompatibility complex (MHC) that are associated with the serologically detected (SD) loci LA and FOUR. Frequently CTLs sensitized to cells from one individual kill target cells of a third party that shares no SD cross-reactivities by serological criteria with the initial stimulating cells. We have studied the possibility that this cross-killing is due to the recognition of non-MHC controlled antigens. Allogeneic tissue from MHC identical siblings is rejected in the absence of immunosuppression, demonstrating the importance of non-MHC antigens. Nevertheless, CTLs sensitized simultaneously with mitomycin-C treated cells from an MHC identical sibling and from an unrelated individual were only able to mediate cytotoxicity on the unrelated's target cells. No significant cytotoxicity was observed in any combination on target cells differing only for non-MHC antigens.