A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release. Target cells were loaded with Calcein-AM and then co-incubated with effector cells. An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media. Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells. Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells. The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets. In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates. The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.