In mitosis and meiosis, cohesion is maintained at the centromere until sister-chromatid separation. Drosophila MEI-S332 is essential for centromeric cohesion in meiosis and contributes to, though is not absolutely required for, cohesion in mitosis. It localizes specifically to centromeres in prometaphase and delocalizes at the metaphase-anaphase transition. In mei-S332 mutants, centromeric sister-chromatid cohesion is lost at anaphase I, giving meiosis II missegregation. MEI-S332 is the founding member of a family of proteins important for chromosome segregation. One likely activity of these proteins is to protect the cohesin subunit Rec8 from cleavage at the metaphase I-anaphase I transition. Although the family members do not show high sequence identity, there are two short stretches of homology, and mutations in conserved residues affect protein function. Here we analyze the cis- and trans-acting factors required for MEI-S332 localization. We find a striking correlation between domains necessary for MEI-S332 centromere localization and conserved regions within the protein family. Drosophila MEI-S332 expressed in human cells localizes to mitotic centromeres, further highlighting this functional conservation. MEI-S332 can localize independently of cohesin, assembling even onto unreplicated chromatids. However, the separase pathway that regulates cohesin dissociation is needed for MEI-S332 delocalization at anaphase.