ABSTRACTThe human pathogenic yeastCandida glabratais the second most commonCandidapathogen afterCandida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA ofC. glabrata(CgCEN), although structurally very similar to that ofSaccharomyces cerevisiae, is not functional inS. cerevisiae. To further examine the structure of theC. glabratainner kinetochore, we isolated severalC. glabratahomologs ofS. cerevisiaeinner kinetochore protein genes, namely, genes for components of the CBF3 complex (Ndc10p, Cep3p, and Ctf13p) and genes for the proteins Mif2p and Cse4p. The amino acid sequence identities of these proteins were 32 to 49% relative toS. cerevisiae. CgNDC10,CgCEP3, andCgCTF13are required for growth inC. glabrataand are specifically found atCgCEN, as demonstrated by chromatin immunoprecipitation experiments. Cross-complementation experiments revealed that the isolated genes, with the exception ofCgCSE4, are species specific and cannot functionally substitute for the corresponding genes inS. cerevisiaedeletion strains. Likewise, theS. cerevisiaeCBF3 genesNDC10,CEP3, andCTF13cannot functionally replace their homologs inC. glabrataCBF3 deletion strains. Two-hybrid analysis revealed several interactions between these proteins, all of which were previously reported for the inner kinetochore proteins ofS. cerevisiae. Our findings indicate that although many of the inner kinetochore components have evolved considerably between the two closely related species, the organization of theC. glabratainner kinetochore is similar to that inS. cerevisiae.