The fission yeast minichromosome loss mutant mcl1-1 was identified in a screen for mutants defective in chromosome segregation. Missegregation of the chromosomes in mcl1-1 mutant cells results from decreased centromeric cohesion between sister chromatids. mcl1+ encodes a beta-transducin-like protein with similarity to a family of eukaryotic proteins that includes Ctf4p from Saccharomyces cerevisiae, sepB from Aspergillus nidulans, and AND-1 from humans. The previously identified fungal members of this protein family also have chromosome segregation defects, but they primarily affect DNA metabolism. Chromosomes from mcl1-1 cells were heterogeneous in size or structure on pulsed-field electrophoresis gels and had elongated heterogeneous telomeres. mcl1-1 was lethal in combination with the DNA checkpoint mutations rad3delta and rad26delta, demonstrating that loss of Mcl1p function leads to DNA damage. mcl1-1 showed an acute sensitivity to DNA damage that affects S-phase progression. It interacts genetically with replication components and causes an S-phase delay when overexpressed. We propose that Mcl1p, like Ctf4p, has a role in regulating DNA replication complexes.