Genetic and biochemical strategies have been used to identify Schizosaccharomyces pombe proteins with roles in centromere function. One protein, identified by both approaches, shows significant homology to the human centromere DNA-binding protein, CENP-B, and is identical to Abp1p (autonomously replicating sequence-binding protein 1) (Murakami, Y., J.A. Huberman, and J. Hurwitz. 1996. Proc. Natl. Acad. Sci. USA. 93:502-507). Abp1p binds in vitro specifically to at least three sites in centromeric central core DNA of S. pombe chromosome II (cc2). Overexpression of abp1 affects mitotic chromosome stability in S. pombe. Although inactivation of the abp1 gene is not lethal, the abp1 null strain displays marked mitotic chromosome instability and a pronounced meiotic defect. The identification of a CENP-B-related centromere DNA-binding protein in S. pombe strongly supports the hypothesis that fission yeast centromeres are structurally and functionally related to the centromeres of higher eukaryotes.