A growing number of clinical vaccine trials involve monitoring of the patient immune response (7,8). One of the main obstacles for comprehensive monitoring is usually a limited amount of patient blood available. It would be beneficial to use methods that allow measurement of different parameters in one test tube. A number of flow cytometric methods to measure cell-mediated cytotoxicity, particularly based on the uptake of propidium iodide (PI), have been offered recently (2–6). Excellent correlation has been found between 7-aminoactinomycin D (7-AAD) inclusion in flow cytometric cytotoxicity assay and the results of the 51Cr release assay, with both fresh and cryopreserved effector cells used. The correlation coefficient R2 was higher than 0.9 in all experiments performed (1). We developed a method based on flow cytometry that would allow us to simultaneously analyze effector cell phenotype and target cell death using the same sample under conditions that ensure reliable discrimination of the target and effector cells. K562 (human myelogenous leukemia cell line; ATCC, Manassas, VA, USA) and C1R.A2 (human plasma leukemia cell line that expresses a transfected genomic clone of HLA-A2.1) were cultured in complete medium (CM) consisting of RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL gentamycin (BioWhittaker, Walkersville, MD, USA). Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of normal human volunteers by buoyant density centrifugation over FicollPaque (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Aliquots of effector cells were cryopreserved in the vapor phase of liquid nitrogen for future use in functional testing and fluorescence-activated cell sorting (FACS) analysis. For cytolytic T lymphocyte DRUG DISCOVERY AND GENOMIC TECHNOLOGIES