Monoclonal antibodies (mAbs) have been derived which identify a target cell antigen involved in human natural killer (NK) cell lysis. The effects of the mAbs on the NK-like cytotoxic activity exhibited by different populations of human cytolytic T cells were examined. The anti-target cell mAbs 1E7 and 18C2 inhibited the lysis of K562 target cells by endogenous NK cells, antigen-specific cytotoxic T lymphocytes (CTL) with NK-like activity, and non-major histocompatibility complex (MHC)-restricted T cells. Cytolysis of K562 target cells was not affected by treatment of the target cells with the anti-class I HLA mAb W6/32. Further, the anti-target cell mAbs had no effect on antigen-specific lysis by the CTL. The mAb W6/32, however, inhibited the antigen-specific killing. Experiments at the single-cell level revealed that the anti-target cell mAbs inhibited the formation of conjugates between the effector cells and K562 tumor cells but had no effect on CTL binding to the antigen-specific target cells. Thus, antigen-specific CTL exhibiting NK-like lytic function appeared to recognize a novel target cell antigen that is distinct from typical MHC antigens and is identical to the target-cell antigen recognized by true NK cells.