The bacteriophage Mu genome contains a strong DNA gyrase binding site (SGS) near its center, and disruption of the SGS by deletion or by insertion results in long delays in replication following induction of the appropriate lysogen. To determine if the central location of the SGS is obligatory for its function in Mu replication, we pursued two lines of investigation. First, fragments of Mu DNA containing the SGS were inserted into various locations in a Mu prophage lacking the central SGS. Replication following induction was restored in all of the lysogens constructed, but the observed rate of replication for different prophages decreased with increasing distance between the new location of the SGS and the center of the genome. We also deleted different lengths of DNA from within the right half of a wild-type prophage, retaining the SGS and displacing it from a central location. Replication rates of the deleted prophages were reduced, with larger deletions resulting in larger reductions. Pairing deletions in the right half of the prophage with a deletion in the left half resulted in substantially higher rates of replication than observed with the right half deletions alone. We conclude that the SGS must be located centrally between the Mu termini for optimal rates of Mu replication. These results are discussed in terms of a model that proposes that the SGS is involved in organizing the topology of supercoiled prophage DNA to assist in synapsis of the Mu termini.