The phage Mu transpositional enhancer has been previously shown to stimulate the initial rate of the Mu DNA strand transfer reaction by a factor of 100. We now show that the Mu enhancer can function in trans on an unlinked DNA molecule. This activity is greatly facilitated by the presence of a free DNA end proximal to the enhancer element. Function of the enhancer in trans does not alter either the requirement for donor DNA supercoiling or for the two Mu ends to be in their proper orientation on the donor plasmid. An important consequence of these findings is that we have been able to evaluate directly the step in the transposition reaction for which the enhancer is required. We show that the role of the enhancer is limited to promoting productive synapsis; efficient strand cleavage can occur in the absence of the enhancer.