The cytotoxic activity of T lymphocytes, natural killer and lymphokine-activated killer cells is usually tested by radioactive assays, which detect the release of cytoplasmic contents after plasma membrane disintegration of dying cells. In contrast to this indirect evaluation of cytotoxicity, we describe here an improved fluorescence assay that is based on the direct quantitative and qualitative flow cytometric analysis of cell damage at a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane and permits distinction between target and effector cells. After 3 h of in vitro incubation, costaining with AnnexinV-FITC (ann-FITC) and propidium iodide (PI) permitted discrimination between vital, early apoptotic and necrotic cells. Data analysis is performed first by gating on PKH-26-positive target cells followed by the analysis of ann-FITC- and PI-positive subpopulations. The percentage of cytotoxicity in the PKH-26-gated cell population is calculated by subtracting non-specific ann-FITC- or PI-positive target cells, measured in appropriate controls without effector cells. Membrane staining of target cells such as primary melanoma cells or leukemic blasts revealed high and stable loading of PKH-26 without altering the viability or the immunogenicity of the cells. Using in vitro-generated antigen-specific cytotoxic T lymphocytes (CTL), we could demonstrate that this flow cytometric assay is sensitive and correlates well with the standard 51Cr release assay. In conclusion, the improved fluorescence assay is a simple and highly reproducible procedure for evaluating the specific cytotoxicity of T cells.