We describe a high-temperature (75 degrees C) transition in the Mu integration complex that causes efficient and true reversal of the integration reaction. A second reversal pathway, first described as 'foldback' reversal for the HIV integrase, was also observed upon disassembly/reassembly of the Mu complex at normal temperatures. Both true and foldback reversal severed only one or the other of the two integrated Mu ends, and each exhibited distinct metal ion specificities. Our results directly implicate an altered transposase configuration in the Mu strand transfer complex that inhibits reversal, thereby regulating the directionality of transposition.