To be able to determine the cytotoxic activity of NK cells or T cells against leukaemic cells in patient samples containing >20% of normal peripheral blood cells, we have developed a four-colour flow cytometric cytotoxicity assay. The assay is based on differential immunostaining of both leukaemic cells and effector cells in combination with propidium iodide (PI). The cytometer is set for measuring the fluorescence of the monoclonal antibody (mAb) bound fluorochromes, with moderate overcompensation of the third and fourth fluorescence signals. PI-positive events were excluded from analysis by their characteristic uncompensated signal on these two detectors. Thus, all four fluorescence ranges can be used for detection of mAb-derived signals and this allows discrimination between various populations contained in effector and target cell samples. The cytotoxic activity in our assay is calculated by the absolute loss of vital leukaemic cells. For this purpose, fluorescent beads are included as an internal standard. When calculating the effector concentrations after coculture, characteristic changes can be seen which yield additional information about the presence of cytotoxic activity and the active effector cell subset. With this assay, we present a versatile tool that combines minimum cell manipulation before coculture with maximum information from a sample. The assay is suitable for the analysis of complex samples with regard to different cell subsets, their decrease or increase, and conjugate formation.