Assays for cytotoxicity of CTLs in vivo using a fluorescent-based dye, 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE), have been established and widely used. On the basis of this experience, we applied it to in vitro assay system and established a simpe, highly sensitive flow cytometric assay for CTL activity. In our assay, specific activities of CTLs could be detected by a reduction in sensitive target cell numbers on single-color histogram plot analysis. By using this assay, we could determine the changes in cytotoxic activity by single amino acid substitution within an epitope peptide. Adherent cells were also used as target cells in this assay by treatment with excess EDTA and trypsin reagents after incubation with effector CTLs. Furthermore, when fluorescent calibration beads were used as a control, we could determine the cytotoxicity of CTLs against tumor cells. The results obtained from our assay were almost consistent with those from the conventional ⁵¹Cr-release assay.Because our assay uses only a stable non-radioactive reagent, CFSE, this assay is safe, inexpensive and extremely easy. These results indicated that this new assay (FACS-CTL assay) would be sufficiently acceptable alternative to classical ⁵¹Cr-release assay.