Mixed lymphocyte reaction (MLR)-activated T cells were analyzed according to the expression of various cell surface markers by the specific cytotoxic T lymphocytes (CTL) generated in the MLR. CTL were found exclusively in a population of MLR-activated T cells that lacked detectable Fc gamma R but that expressed a surface antigen recognized by the 4F2 monoclonal antibody. In contrast, CTL were found in both the Ia-positive and Ia-negative cells after MLR activation. Thus, the specific CTL generated in the allogeneic MLR can be identified and isolated by virtue of the expression of a particular cell surface marker.