The increasing interest in programmed cell death has created the need to measure apoptosis in complex cell systems. We have combined the use of fluorescent antibodies with the Hoechst 33342/propidium iodide system in order to quantitate programmed cell death in fractions of heterogenous cell populations. Here we describe the analysis of T-cell apoptosis after ligation of the T-cell antigen receptor by superantigen in vitro and ex vivo. This technique can separate cells according to seven parameters, fluorescence caused by FITC, PE, allophycocyanin, incorporation of Hoechst 33342, PI, forward scatter, and side scatter, and it allows determination of elevated Hoechst 33342 uptake in less than 10% of the cell population.