Molecular genetic analysis of the yeast Ebp2 protein has revealed that it is an essential, nucleolar protein that functions in the rRNA biosynthesis pathway. Temperature-sensitive ebp2-1 mutants are defective in the processing of the 27 SA precursor rRNA, and the point substitutions that disrupt this activity cluster towards the central, more highly conserved region of the Ebp2 protein. We report here that other ebp2 mutants exhibit deficiencies associated with defects in chromosome segregation. Yeast cells bearing a 50 amino acid C-terminal truncation allele (ebp2ΔC50) display a slow-growth phenotype and exhibit an increased percentage of cells with the nucleus positioned at the bud neck. The ebp2-1 and ebp2ΔC50 alleles genetically complement each other, and ebp2ΔC50 mutants exhibit nuclear division defects that are distinct from the rRNA biosynthesis-related phenotypes of ebp2-1 mutants. Cytological and FACS analysis of the ebp2ΔC50 deletion mutants indicate that the chromosome segregation related activities of the Ebp2 protein are monitored by Mad2p, a mitotic checkpoint protein. The finding that yeast Ebp2p functions in nuclear division is consistent with the growing body of evidence that supports the role that human EBP2 plays in chromosome segregation. Copyright © 2004 John Wiley & Sons, Ltd.