Chromosomes segregate at mitosis along microtubules attached to the kinetochore, an organelle that assembles at the centromere. Despite major advances in defining molecular components of the yeast segregation apparatus, including discrete centromere sequences and proteins of the kinetochore, relatively little is known of corresponding elements in more complex eukaryotes. We show here that human CENP-C, a human autoantigen previously localized to the kinetochore, assembles at centromeres of divergent species, and that the specificity of this targeting is maintained by an inherent destruction mechanism that prevents the accumulation of CENP-C and toxicity of mistargeted CENP-C. The N-terminus of CENP-C is not only required for CENP-C destruction but renders unstable proteins that otherwise possess long half-lives. The conserved targeting of CENP-C is underscored by the discovery of significant homology between regions of CENP-C and Mif2, a protein of Saccharomyces cerevisiae required for the correct segregation of chromosomes. Mutations in the Mif2 homology domain of CENP-C impair the ability of CENP-C to assemble at the kinetochore. Together, these data indicate that essential elements of the chromosome segregation apparatus are conserved in eukaryotes.