Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy-1 dim thymocytes were included in the dye dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin. However, the dyes SYTO-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser. © 1995 Wiley-Liss, Inc.