Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.