Abstract: Monitoring Editor: Kerry Bloom Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of greater than 65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly (Hill and Bloom, 1987, 1989). Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1-phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant, Cse4, and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.