Upon catalyzing strand transfer, the Mu transposase (MuA) remains tightly bound to the resulting transposition intermediate, the strand transfer complex (STC), and poses an impediment to host replication proteins. Additional host factors, which can be resolved into two fractions (Mu Replication Factor alpha and beta; MRF alpha and MRF beta), are required to disassemble the MuA complex and initiate DNA synthesis. MRF alpha modifies the protein content of the STC, removing MuA from the DNA in the process. The MRF beta promotes initiation of the Mu DNA synthesis on the STC altered by the MRF alpha. These host factors cannot promote initiation of Mu DNA synthesis if the STC is damaged by partial proteolysis. Moreover, the mutant protein MuA211 cannot be removed from the STC by MRF alpha, blocking initiation of DNA synthesis. These results indicate that MuA in the STC plays a critical function in beginning a sequence of events leading to the establishment of a Mu replication fork.