During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defective crossing over causes infertility, miscarriage and congenital disease. Each pair of chromosomes attains at least one crossover via the formation and biased resolution of recombination intermediates known as double Holliday junctions 2,3 . A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 4 . The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers 5-10 , but the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp PCNA is important for crossover-biased resolution. In vitro assays with human enzymes show that PCNA and its loader RFC are sufficient to activate the MutLγ endonuclease. MutLγ is further stimulated by a co-dependent activity of the pro-crossover factors EXO1 and MutSγ, the latter of which binds Holliday junctions 11 . MutLγ also binds various branched DNAs, including Holliday junctions, but does not show canonical resolvase activity, implying that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, RFC facilitates MutLγ-dependent crossing over in budding yeast. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair 12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.