The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.