The bacteriophage P1 Cre recombinase catalyzes site-specific recombination between 34-base-pair loxP sequences in a variety of topological contexts. This reaction is widely used to manipulate DNA molecules in applications ranging from benchtop cloning to genome modifications in transgenic animals. Despite the simple, highly symmetric nature of the Cre-loxP system, there is strong evidence that the reaction is asymmetric; the 'bottom' strands in the recombining loxP sites are preferentially exchanged before the 'top' strands. Here, we address the mechanistic basis for ordered strand exchange in the Cre-loxP recombination pathway. Using suicide substrates containing 5'-bridging phosphorothioate linkages at both cleavage sites, fluorescence resonance energy transfer between synapsed loxP sites and a Cre mutant that can cleave the bridging phosphorothioate linkage but not a normal phosphodiester linkage, we showed that preferential formation of a specific synaptic complex between loxP sites imposes ordered strand exchange during recombination and that synapsis stimulates cleavage of loxP sites.