Two out of four long-term murine allospecific cytotoxic T lymphocyte (CTL) clones tested could develop high levels of cytotoxicity against syngeneic target cells when cultured under appropriate conditions. All CTL clones maintained strict allospecificity so long as they were cultured with both appropriate allogeneic stimulator cells and growth factor (supernatant from secondary mixed lymphocyte cultures). In two of the clones, syngeneic reactivity rapidly developed when the allogeneic stimulator cells were replaced with syngeneic or third party stimulator cells, and when the supernatant from EL4 thymoma cells stimulated with phorbol ester was used as growth factor. In addition to killing the appropriate allogeneic target, clones with syngeneic reactivity could kill both syngeneic C57BL/6 targets and H-2-congenic BALB.B targets but not third party unrelated targets, suggesting that the self structure recognized was coded for within the major histocompatibility complex. Such clones did not kill the natural killer (NK) target YAC. The results obtained from cold target inhibition and from subcloning at limiting dilution of clones with syngeneic reactivity suggested that both allogeneic and syngeneic reactivity could be expressed by the same individual cell in the CTL clone. The specificity for syngeneic H-2 as opposed to third party H-2 and NK-sensitive target cells, and the observation that both allospecific and syngeneic killing could be partially blocked by anti-Lyt-2 antibody treatment of the CTL, strongly suggested that different recognition structures are involved in CTL-mediated syngeneic cytotoxicity and NK cytotoxicity.