Tn4430 is a distinctive transposon of the Tn3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro. Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented.