The 51Cr release assay has been the method of choice in analyzing natural killer cell (NK) function. Previous FCM cytotoxicity assays of NK activity have had numerous disadvantages that discouraged clinicians from attempting to evaluate NK function by flow cytometry. We demonstrate the effectiveness of using PKH-26, a stable membrane dye, to label the K562 target cells and propidium iodide intercalation into killed target cell DNA to determine the percentage of target cells killed by effector NK cells from the peripheral blood or bone marrow. This method compares favorably with the 51Cr release assay and is quicker and easier to perform. The percentage of cytotoxicity of NK cells (CD3- CD56+ and/or CD16+) from 10 normal subjects and 10 HIV-infected children are reported to demonstrate the feasibility of studying NK function in clinical populations by FCM. The potentiation of cytolysis by alpha-interferon and interleukin 2 in vitro was also compared between these two study groups. In addition, a patient whose leukemic blasts expressed CD56+ was also studied for NK activity using this flow cytometric assay. The benefits of using this flow cytometric approach to clinically assess NK function are discussed.