Essential meiotic endonuclease 1 (Eme1), a member of the Eme1/Mms4 family, associates with Mus81 to constitute a heterodimeric endonuclease that has been implicated in mitotic and meiotic recombination in eukaryotes. The Mus81-Eme1 complex cleaves branched DNA structures, especially those arising during stalled DNA replication such as replication forks and 3' DNA flaps. When purified from yeast, this complex cleaves synthetic Holliday junctions into linear duplex DNA. These findings provide compelling evidence that Mus81-Eme1 complexes are essential elements of the eukaryotic nuclear Holliday junction resolvase. Eme1 may also be required in mitosis for the processing of collapsed replication forks. Eme1 is typically localized to the nucleolus and is recruited to regions of DNA damage in S phase cells.