The aim of the present study was to define the cell lineage of mixed lymphocyte culture (MLC)-induced natural killer (NK) effector cells. Human MLC cells were plated under limiting microculture conditions in the presence of irradiated spleen cells and interleukin 2-containing supernatant. After 18 days, microcultures were scored for proliferation and for cytolytic activity against specific lymphoblasts and NK-sensitive K562 target cells. About 1 in 7 and 1 in 5 proliferating microcultures had specific or NK-like cytolytic activity, respectively. Moreover, several microcultures exhibited dual (specific and NK-like) cytolytic activity, even when they had been established at relatively low numbers of responding cells/well (0.5-0.25) to ensure a high probability of monoclonality. Direct evidence for the existence of cytolytic effector cells with dual activity was achieved by using clones derived from single MLC T cells by micromanipulation. Out of 26 cytolytic clones so derived, 16 exhibited specific cytolytic activity, whereas 22 lysed K562 target cells. More interestingly, 12 of these 26 clones were active against both types of target cells. Only one of these clones was able to lyse autologous or unrelated target cells. In contrast, all such clones lysed the NK-sensitive cell lines G11, MOLT-4, Raji, Daudi, Chang and T-24. Addition of saturating amounts of B9-4 monoclonal antibody in the lytic assays resulted in the inhibition of the specific cytolysis, but not the NK-like activity of clones with dual cytolytic activity. It thus appears that (a) alloreactive cytotoxic T lymphocytes can mediate both specific and NK-like cytolysis and (b) two independent recognition structures are involved in this dual activity.