The MuA and MuB proteins collaborate to mediate efficient transposition of the phage Mu genome into many DNA target sites. MuA (the transposase) carries out all the DNA cleavage and joining steps. MuB stimulates strand transfer by activating the MuA-donor DNA complex through direct protein-protein contact. The C-terminal domain of MuA is required for this MuA-MuB interaction. Activation of strand transfer occurs irrespective of whether MuB is bound to target DNA. When high levels of MuA generate a pool of free MuB (not bound to DNA) or when chemical modification of MuB impairs its ability to bind DNA, MuB still stimulates strand transfer. However, under these conditions, intramolecular target sites are used exclusively because of their close proximity to the MuA-MuB-donor DNA complex.