A flow cytometry-based assay for analyzing cytotoxic T lymphocyte (CTL) activity is presented. This new approach is characterized by easy handling, the generation of highly reproducible data sets and is not dependent on the use of radioactivity. Before exposure to primed CTL effector cells the target cells were labeled with the green fluorescent dye DiO18(3) which is incorporated stably into the cell membrane. After a 4-h incubation period, samples were counterstained with the red fluorescent nuclear dye propidium iodide in order to permit discrimination between live and dead cells within both cell populations. The assay has been used to quantitate CTL effector activity against allogeneic lymphoblasts. Results derived from this novel flow cytometry assay show an excellent correlation (r = 0.988) with data obtained using the standard 51chromium release assay. An additional advantage of the assay is that freshly prepared splenocytes may be used as target cells because culturing and activation of target cells is no longer required. The results demonstrate that the fluorescent dyes DiO18(3) and propidium iodide in combination with flow cytometry permit accurate analysis of cytotoxic T cell activity.