Apoptosis occurs under many different physiological and pathological conditions, and it reflects a genetically encoded suicide program that can be triggered by different stimuli in susceptible cells. We adapted a flow cytometric assay for the detection of apoptosis based on differential staining of viable cells with two different DNA binding dyes, propidium iodide (PI) and Hoechst 33342 (Ho342). Apoptosis was induced in different cell lines by gamma irradiation, an anti-FAS monoclonal antibody, or ganciclovir in Herpes simplex virus-thymidine kinase-expressing cells. We could identify three different populations that appeared sequentially after the induction of apoptosis. Cells corresponding to these populations were sorted and assessed for evidence of apoptosis as determined by alterations of nuclear morphology and detection of endonucleolytic activity. This analysis revealed a PI- population with subtle apoptotic changes and increased Ho342 fluorescence compared with untreated cells. Extensive apoptotic alterations were observed in a PI+ population that increased over time following the induction of apoptosis. A third population was characterized by an intermediate intensity of PI fluorescence and decreased Ho342 fluorescence compared with the other populations. This population appeared late after treatment and consisted of apoptotic bodies. Taken together, these data suggest that distinct stages of apoptosis can be identified by differential staining of cells with Ho342 and PI. This assay should be useful for the detection and further characterization of cells at different stages in the apoptotic process.