Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for t he enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II a that has phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduces the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar, but stronger effect was seen when the homologous mutation (Tyr28AEPhe) was introduced in yeast TOP2. The ATPase activities of both hTOP2a (Tyr50AEPhe) and yTOP2 (Tyr28AEPhe) were resistant to both bisdioxopiperazines, and also to ATPase inhibitor sodium orthovanadate. Vanadate, like bisdioxopiperazines, traps the enzyme in a salt-stable closed conformation termed the closed clamp that can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme, and that this difference in stability may lead to a loss of DNA stimulated ATPase. We suggest that the DNA stimulated ATPase of topoisomerase II is intimately connected with steps occurring while the N-terminal domain of the enzyme is dimerized.