A rapid, gentle, and sensitive method for quantification of cells undergoing apoptosis is presented. The method allows the simultaneous determination of dual-color cell surface immunofluorescence. Cells are stained for 7 min with the vital dye Hoechst 33342 (HO342) for identification of live and apoptotic cells. 7-amino-actinomycin D (7-AAD) is added to distinguish cells that have lost membrane integrity from apoptotic and live cells. Due to its spectral properties 7-AAD can be utilized on cells that are dual-surface labelled with fluoresceinisothiocyanate (FITC) and phycoerythrin (PE). The value of the method is demonstrated on human thymocytes, which constitutively undergo programmed cell death and which show an increase in the rate of apoptosis after exposure to the glucocorticoid dexamethasone (DEX). Vital staining with HO342 permits earlier detection of apoptotic changes compared to a staining technique in which cells are treated with a hypotonic citrate solution containing propidium iodide (PI) and the apoptotic cells are represented in a hypodiploid, “sub-G1” peak. The HO342/7AAD method may be particularly applicable to studies of programmed cell death in cells in which DNA fragmentation is difficult to detect by decreased DNA stainability. © 1994 Wiley-Liss, Inc.