The specificity of antigen recognition by in vitro sensitized human cytotoxic T lymphocytes (CTLs) has been studied using a sensitive cell-mediated lympholysis (CML) assay. Frequently, high levels of cytotoxicity are observed on third-party targets unrelated to sensitizing or responding cells; however, no cytotoxicity differing significantly from zero has been observed on targets autologous to the responding CTLs. This "cross-killing" of third-party target cells has been observed when stimulating and third-party cells bear no cross-reacting serologically defined (SD) antigens, thought to be the target antigens recognized by CTLs. CML-blocking studies, using unlabeled normal human lymphocytes to inhibit 51Cr release from radiolabeled target cells, have shown that cross-killing, even in the absence of shared SD determinants, results from CTLs recognizing antigens shared by the third-party targets and the initial stimulating population. Furthermore, these antigens have been mapped to the major histocompatibility complex (MHC). The ability of human CTLs to specifically recognize MHC-controlled antigens not detected serologically suggests that SD antigens may be recognized differently by alloantisera and CTLs, or that MHC antigens other than SD may be the targets of CTLs in CML.