The location of the cis-acting DNA sequences that direct the assembly of the mammalian kinetochore is not known. A variety of circumstantial evidence, however, has led to the widespread belief that they are present throughout the kinetochore including the kinetochore outer plate. To investigate this question directly, we have used two independent methods to localize DNA in and around the mammalian kinetochore. Both methods fail to reveal DNA in the outer kinetochore plate, finding instead that the outer-most detectable DNA in the centromere is located in the inner kinetochore plate. Our results imply that the outer kinetochore plate is primarily a proteinaceous structure. It is thus unlikely that fibers observed in the outer plate correspond to chromatin, as previously assumed. Our observations suggest that current models of kinetochore structure may need to be reconsidered.

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