Cytotoxic cells are produced in an autologous mixed leukocyte reaction (AMLR). At 1 wk in culture the AMLR killers are mainly IgG Fc- cells and can kill autologous lymphoblastoid cell lines and Raji and Daudi targets that are usually resistant to natural killer cell (NK) lysis. To define the phenotype of these cells, we have used complement (C')-mediated lysis with monoclonal antibodies (MAb). AMLR killer activity was virtually eliminated by treatment with C' and 9.6 or 4F2, but the cytotoxic cells did not express NK-specific antigens, OKM1 and Leu-7, nor cytolytic T lymphocyte-specific antigens, 9.3 and OKT8. None of the 10 MAb used could significantly block cytotoxicity at the final concentration of 1.5 mcg/ml which is generally sufficient to inhibit CTL. The majority of cells at 1 wk in AMLR cultures stained with T cell activation antigens Ia and 4F2; AMLR killing was proportional to the percentage of 4F2+ cells but unrelated to the expression of Ia antigen.