Little is known about the precise ways in which monocytes and macrophages recognize tumor cells and how they exert their cytolytic and/or cytostatic effects. By a functional, morphologic, and flow-cytometric approach, we have studied monocyte/macrophage- and cytokine-mediated cytotoxicity against U937 cells, a human histiocytic lymphoma cell line. A rapid decrease in cell viability of U937 cells (MTT assay) could be observed at an effector-to-target cell (E:T) ratio of 10 in the presence of interferon (IFN)-gamma-activated monocytes. Light and electron microscopic examination showed the characteristic features of apoptosis of U937 cells after incubation with either monocytes or tumor necrosis factor (TNF)-alpha. TNF-alpha-induced apoptosis (10(4) U/mL) as measured by multiparameter flow cytometry (propidium iodide [PI]) paralleled the functional decrease in cell viability (MTT assay) of 20 +/- 3% after 24 hours up to a maximum of 50 +/- 4% after 48 hours. Apoptosis could be confirmed by the detection of DNA degradation into multiples of 200-bp subunits by agarose gel electrophoresis. After prolonged incubation times, monocyte-mediated leukemic cell death could be quantified as apoptosis by flow cytometry, whereas no decrease in net cell viability of tumor cells relative to the initial cell number could be observed by MTT spectrophotometry. In conclusion, our data provide evidence that apoptosis is the major mode of TNF-alpha-dependent monocyte-mediated cytotoxicity against U937 cells. Furthermore, multiparameter flow-cytometric analysis offers a sensitive method to quantify cytokine- and cell-induced apoptosis in leukemia.