For a cell to complete DNA replication, every link between the Watson-Crick strands must be removed by topoisomerases. Previously, we reported that the inhibition of topoisomerase IV (topo IV) leads to the accumulation of catenated plasmid replicons to a steady-state level of approximately 10%. Using pulse labeling with [3H]thymidine in Escherichia coli, we have found that in the absence of topo IV activity, nearly all newly synthesized plasmid DNA is catenated. Pulse-chase protocols revealed that catenanes are metabolized even in the absence of topo IV and that the residual turnover is carried out by DNA gyrase at a rate of approximately 0.01/sec. Using extremely short pulse-labeling times, we identified significant amounts of replication catenanes in wild-type cells. The rate of catenane unlinking in wild-type cells by the combined activities of topo IV and DNA gyrase was approximately 1/sec. Therefore, gyrase is 100-fold less efficient than topo IV in plasmid replicon decatenation in vivo. This may explain why a fully functional gyrase cannot prevent the catenation of newly synthesized plasmid DNA and the partition phenotype of topo IV mutants. We conclude that catenanes are kinetic intermediates in DNA replication and that the essential role of topo IV is to unlink daughter replicons.