The two chemical steps of phage Mu transpositional recombination, donor DNA cleavage and strand transfer, take place within higher order protein-DNA complexes called transpososomes. At the core of these complexes is a tetramer of MuA (the transposase), bound to the two ends of the Mu genome. While transpososome assembly normally requires a number of cofactors, under certain conditions only MuA and a short DNA fragment are required. DNA requirements for this process, as well as the stability and activity of the ensuing complexes, were established. The divalent cation normally required for assembly of the stable complex could be omitted if the substrate was prenicked, if the flanking DNA was very short or if the two flanking strands were non-complementary. The presence of a single nucleotide beyond the Mu genome end on the non-cut strand was critical for transpososome stability. Donor cleavage additionally required at least two flanking nucleotides on the strand to be cleaved. The flanking DNA double helix was destabilized, implying distortion of the DNA near the active site. Although donor cleavage required Mg2+, strand transfer took place in the presence of Ca2+ as well, suggesting a conformational difference in the active site for the two chemical steps.