The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)