Epipodophyllotoxins are an important new class of anticancer agents which include the compounds VM-26 (teniposide) and VP-16 (etoposide). The mechanism of action of these drugs appears to involve production of DNA single- and double-strand breaks by virtue of a temperature-sensitive interaction between drug and a heat-labile intranuclear component. Type II topoisomerase is the likely intracellular target for the DNA strand-breaking effects of the epipodophyllotoxins. Both VM-26 and VP-16 stimulate site-specific DNA cleavage by a highly purified calf thymus type II topoisomerase. VM-26 is 5- to 10-fold more potent than VP-16 in this assay, a difference that is also seen when DNA strand breaks are assayed in isolated nuclei of mouse leukemia cells following drug exposure. A similar potency difference exists with respect to cytotoxicity. Equilibrium dialysis experiments using [3H]VP-16 indicate that the drug does not bind to DNA. Epipodophyllotoxins evidently exert their anti-cancer effects by poisoning type II topoisomerase without binding to DNA. In this regard, their actions may be analogous to those of nalidixic acid in bacteria.