We identified a novel essential centromere protein, CENP-I, which shows sequence similarity with fission yeast Mis6 protein, and we showed that CENP-I is a constitutive component of the centromere that colocalizes with CENP-A, -C, and -H throughout the cell cycle in vertebrate cells. To determine the precise function of CENP-I, we examined its role in centromere function by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-I, cells arrested at prometaphase with misaligned chromosomes for long periods of time. Eventually, cells exited mitosis without undergoing cytokinesis. Immunocytochemical analysis of CENP-I-deficient cells demonstrated that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere.