Topoisomerase II is the target for two broad groups of clinically relevant drugs. Members of these groups are classically defined by their ability to enhance enzyme-mediated DNA cleavage (such as etoposide and m-AMSA) or to inhibit enzyme-catalyzed ATP hydrolysis (such as novobiocin). The above notwithstanding, little is known concerning the interactions of drugs in either mechanistic class with the topoisomerase II-DNA complex. In order to further delineate the mechanism of drug action, the effects of several topoisomerase II-targeted agents on the DNA cleavage and ATP hydrolysis steps of the enzyme's catalytic cycle were determined. Of the drugs examined (genistein, quercetin, quercitrin, etoposide, m-AMSA, CP-115,953, and novobiocin), only novobiocin was unable to enhance topoisomerase II-mediated DNA cleavage. Moreover, with the exception of etoposide, all of the drugs were found to inhibit enzyme-catalyzed ATP hydrolysis. This latter finding undercuts the common assumption that DNA cleavage-enhancing drugs are specific for the cleavage/religation activity of topoisomerase II. Finally, by utilizing a series of competition experiments that took advantage of mechanistic differences between drug classes, it was possible to functionally define drug interaction domains on the eukaryotic type II enzyme. Results of this novel approach indicate that the interaction domain for novobiocin on topoisomerase II is distinct from those of the DNA cleavage-enhancing drugs.