Histones are fundamental structural components of chromatin and are expected to play important roles in chromosome dynamics. Here, we present direct evidence that Cse4p, a histone H3 variant, is a structural component of the core centromere of S. cerevisiae. In histone H4 and Cse4p mutants, the core centromere chromatin structure is disrupted at restrictive temperature. Overexpression of Cse4p suppresses this defect in the H4 mutant, implying that the two proteins act together in centromere structure. We show by chromatin immunoprecipitation experiments that Cse4p is specifically cross-linked to centromeric DNA. Furthermore, by immunofluorescence microscopy, Cse4p is found in discrete foci consistent with that expected for centromeres. These results suggest the kinetochore is assembled on a specialized centromeric nucleosome containing Cse4p.