Type II DNA topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. The steady-state fraction of knotted or catenated DNA molecules produced by prokaryotic and eukaryotic type II topoisomerases was found to be as much as 80 times lower than at thermodynamic equilibrium. These enzymes also yielded a tighter distribution of linking number topoisomers than at equilibrium. Thus, topoisomerases do not merely catalyze passage of randomly juxtaposed DNA segments but control a global property of DNA, its topology. The results imply that type II topoisomerases use the energy of adenosine triphosphate hydrolysis to preferentially remove the topological links that provide barriers to DNA segregation.