The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly, but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A, a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 and that resembles the yeast centromeric component CSE4. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes, our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in α-satellite DNA. However, the association of CENP-A with neocentromeres lacking detectable α-satellite DNA, and the lack of CENP-A association with α-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.