Bisbenzimidazoles (Hoechst 33342 and Hoechst 33258) are cell permeable, adenine-thymine binding fluorescent dyes used to stain deoxyribonucleic acid (DNA) during the evaluation of cell cycle, induction of apoptosis by various ligands and cell viability by flow cytometry. These dyes inhibit topoisomerase I activity in vitro, like camptothecin. In this study, Hoechst 33342 is shown to induce apoptosis at concentration of 10 micrograms/mL or greater after 3 hours incubation in Dulbecco's Modified Eagle Medium characterized by rounded cell morphology, half-moon nuclei with condensed chromatin and a DNA fragmentation ladder of 180 base pair multiples. Hoechst 33258 at the same molarity or seven times greater molarity did not induce apoptosis. If the BC3H-1 myocytes were incubated in RPMI-1640 media, two times the concentration of Hoechst 33342 (20 micrograms/mL) was required to initiate apoptosis. Staining of unfixed cells with Hoechst 33342 may induce apoptosis in the absence of ligands. Therefore, Hoechst 33342 concentration and staining interval should be tested before ligands which may induce apoptosis are evaluated.