In order to utilize a newly available scanning microfluorimeter for lymphocyte-mediated cytotoxicity assays, a number of commercially available fluorescent dyes were compared for their suitability as target cell markers. One of them, bis-carboxyethyl-carboxyfluorescein (BCEFCF), was useful for assays with about 10(4) target cells and showed substantially less spontaneous leakage than other fluorescein derivatives, while still leaking more rapidly than 51Cr. For short cytotoxicity incubations (less than 2 h) with cytotoxic T lymphocytes (CTL), the corrected percentage BCECF release into the supernatant parallels that of 51Cr. For 4 h assays cytotoxicity could be quantitated by measuring the BCECF retained by target cells. Using human CTL and natural killer (NK) cells as effectors, with a variety of lymphoid cells and fibroblasts as targets in 4 h assays, the BCECF retention technique was found to give cytotoxicity values comparable to the 51Cr release assay. Cytotoxicity assays measuring BCECF fluorescence in microtiter wells with the scanning microfluorimeter offer advantages of safety, economy, and processing time compared with the 51Cr release assay.