Topoisomerase IV (Topo IV), encoded by parC and parE, is required for partition of the daughter chromosomes in Escherichia coli. This enzyme is likely responsible for decatenating the linked daughter chromosomes after replication. In this report, we have examined the action of Topo IV in both pBR322 and oriC DNA replication reconstituted in vitro with purified proteins. Gyrase fails to decatenate the linked daughter molecules under any condition in the oriC system and at physiological salt concentrations in the pBR322 system, whereas Topo IV stimulates generation of monomer product DNA by 7- to 10-fold. Topo IV-catalyzed decatenation of isolated multiply linked DNA dimers was relatively insensitive to salt; it proceeded at 14% of the maximal rate even in the presence of 800 mM potassium glutamate. In contrast, decatenation in vitro by gyrase was inhibited completely under these conditions. Pulse-chase analysis indicated that Topo IV-catalyzed resolution of linked daughter DNA molecules occurred prior to completion of DNA replication, such that multiply linked daughter molecules did not arise. These results suggest that during DNA replication, gyrase acts primarily to relieve accumulated positive supercoiling and Topo IV acts to segregate the daughter chromosomes.