Multiparameter flow cytometry and cell sorting were used to examine the process of apoptosis after activation of murine resting T cells with immobilized anti-CD3. Activated T cells treated with Hoechst 33342 (HO-33342) and analyzed by flow cytometry showed two major cell populations of high and low fluorescence. These populations were sorted and the DNA extracted and subjected to electrophoresis. Electrophoresis of DNA extracted from T cells showing a low level of HO-33342 fluorescence (HO-Low) resulted in a typical ladder pattern characteristic of internucleosomal DNA degradation associated with apoptosis, whereas the cellular DNA of the cells showing a high level of fluorescence (HO-High) showed a narrow high molecular weight band. Multiparameter analysis further indicated that cells with HO-High characteristics possessed corresponding high-FSC/low-SSC properties, whereas HO-Low cells formed a cluster of low-FSC/high-SSC cells. Analysis of the DNA extracted from cells sorted on the basis of scatter properties alone confirmed that the low-FSC/high-SSC population contained the apoptotic cells and that the high-FSC/low-SSC population was comprised of viable cells. This methodology allowed us to determine the percentage of apoptotic cells following anti-CD3 activation at various time points and to discriminate them from those in cell cycle. We could further quantitate the number of apoptotic versus viable CD4+ and CD8+ cells in the cell cycle. © 1993 Wiley-Liss, Inc.