The Holliday junction is a key DNA intermediate in the process of genetic recombination. It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices. RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules. We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction. We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system. We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage.