This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single- and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.