The strand cleavage and strand transfer reactions of Mu DNA transposition require structural/catalytic contributions from separate polypeptide domains of individual transposase (MuA) monomers within the functional MuA tetramer. Based on catalytic complementation between two inactive MuA variants, we have derived certain rules by which the physical location of a MuA monomer within the transposition complex specifies its role in DNA breakage and transfer. During strand transfer, MuA monomers contributing domain II to the reaction occupy R1 (the subsite proximal to the strand-transferred nucleotide), while those contributing domain IIIalpha occupy R2. The positions of the monomers contributing these two domains appear to be reversed during DNA cleavage.