Reconstitution of the activation of the MLH1-MLH3 endonuclease shows how crossovers are formed during meiosis. During prophase of the first meiotic division, cells deliberately break their DNA(1). These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes(2). A pathway that depends on the MLH1-MLH3 (MutL gamma) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism(3-7). Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutS gamma), which supports crossing over(8), binds branched recombination intermediates and associates with MutL gamma, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutS gamma directly stimulates DNA cleavage by the MutL gamma endonuclease. MutL gamma activity is further stimulated by EXO1, but only when MutS gamma is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over.Saccharomyces cerevisiaestrains in which MutL gamma cannot interact with PCNA present defects in forming crossovers. Finally, the MutL gamma-MutS gamma-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points(9). As DNA nicking by MutL gamma depends on its co-factors, the asymmetric distribution of MutS gamma and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutL gamma nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes(4).