Peripheral blood leukemia cells from patients with acute monoblastic leukemia (AMoL) were tested for killer cell activity against target cells that detected natural killer cell-mediated or monocyte-mediated spontaneous cytotoxicity. The fibrosarcoma cell line Wehi 164, pretreated with actinomycin D to induce susceptibility to lysis, specifically detects the activity of unstimulated human monocytes. In four of six cases of AMoL, high killer cell activity could be measured against this target. In three of these four cases, the killer cell activity could be assigned exclusively to the leukemic clone, based on the high leukocyte counts and the resultant dilution of normal cells, as evidenced by marker and by functional analysis. While leukemic cells with killer cell activity against Wehi 164 contained 34% to 45% cells that were positive for binding of the 63D3 monoclonal antibody, the two leukemic samples without killer cell activity contained only 1% and 12% 63D3-positive cells. Cell sorting of 63D3-positive and -negative cells from two leukemias with killer cell activity demonstrated that the killer cell activity was restricted to the 63D3-positive fraction of AMoL cells. These data demonstrate that monoblastic leukemia cells can be potent killer cells and that killing activity is linked to the 63D3- defined cell surface molecule.