Activation of human peripheral blood lymphocytes (PBL) with the mitogenic monoclonal antibody (MoAb) K46M, which recognizes 1-5% of PBL, resulted in the expansion of cells with cytolytic activity. Thus, after culture of the activated lymphocytes in medium containing interleukin 2 (IL-2), they lysed a variety of cultured cell lines. The majority of the activated lymphocytes reacted with MoAb to CD8, CD3, and to the T cell antigen receptor heterodimer (Ti) but not with antibodies to antigens expressed on natural killer (NK) cells. The cytotoxicity was not inhibited by MoAb to CD3 or Ti. However, the killing of K562, but not of other cell lines, was enhanced by three to four times in the presence of anti-Ti antibodies. Anti-CD3 or other control antibodies had no effect. Cold target inhibition experiments indicated that the cytolytic lymphocytes recognized closely related structures on the target cells. Phenotypically and functionally similar effector cells emerged after activation of PBL with the anti-CD3 MoAb OKT3. Taken together, the results indicate that activation of PBL with MoAb K46M induces cytotoxic cells that differ from classical NK cells but that resemble mature cytotoxic T lymphocytes (CTL). However, unlike CTL, cytotoxicity was not MHC-restricted and the conventional T-cell receptor complex (CD3/Ti) appeared not to be involved in target cell recognition and cytolysis.