The RuvAB and RuvC enzymes of Escherichia coli define a molecular pathway for the resolution of Holliday intermediates in recombination and DNA repair. They bind specifically to Holliday junctions, and catalyse their branch migration and cleavage, respectively. In a RuvA(B)-junction complex, the Holliday structure is held in an open (square planar) configuration on the concave surface of a 4-fold symmetrical tetramer of RuvA, whereas in a RuvC-junction complex it is folded in an alternative arrangement as part of the cleavage reaction. Genetic studies have shown that the activity of RuvC in vivo depends on RuvAB, which suggests that the two enzymes act in concert, with junction cleavage by RuvC following from branch migration by RuvAB. We have investigated how RuvC can take over a junction from RuvAB to cleave the DNA. We show that RuvA inhibits junction cleavage by RuvC, probably by sandwiching the junction between two tetramers. The extent of inhibition depends on the reaction kinetics of RuvA binding relative to RuvC binding and cleavage. The presence of RuvB and the concentration of Mg2+ both have a significant effect on cleavage in the presence of RuvA. However, a novel protein-DNA complex can be formed when junction DNA is incubated with both RuvA and RuvC. Its mobility is consistent with a RuvC dimer binding to a junction held in an open configuration on the surface of a RuvA tetramer. We suggest that this arrangement provides RuvC with the means to scan the junction during the RuvAB-mediated branch migration reaction for DNA sequences that it can cleave. We further suggest that recognition of the target may provide a trigger for dissociating RuvA, allowing the junction to be folded and cleaved by RuvC.