Knotted DNA has potentially devastating effects on cells. By using two site-specific recombination systems, we tied all biologically significant simple DNA knots in Escherichia coli. When topoisomerase IV activity was blocked, either with a drug or in a temperature-sensitive mutant, the knotted recombination intermediates accumulated whether or not gyrase was active. In contrast to its decatenation activity, which is strongly affected by DNA supercoiling, topoisomerase IV unknotted DNA independently of supercoiling. This differential supercoiling effect held true regardless of the relative sizes of the catenanes and knots. Finally, topoisomerase IV unknotted DNA equally well when DNA replication was blocked with hydroxyurea. We conclude that topoisomerase IV, not gyrase, unknots DNA and that it is able to access DNA in the cell freely. With these results, it is now possible to assign completely the topological roles of the topoisomerases in E. coli. It is clear that the topoisomerases in the cell have distinct and nonoverlapping roles. Consequently, our results suggest limitations in assigning a physiological function to a protein based upon sequence similarity or even upon in vitro biochemical activity.