We have used in vitro motility assays to investigate the mechanism of kinetochore function in the budding yeast Saccharomyces cerevisiae. Functional centromeric DNA plus a tripartite centromere binding protein complex, CBF3, was found to be necessary but not sufficient for in vitro kinetochore activity. A fourth required component was identified as the motor protein Kar3p, a previously reported yeast kinesin known to be involved in karyogamy and mitosis. Our data support genetic evidence suggesting that Kar3p is a kinetochore-associated motor and imply that CBF3 plays a regulatory role in kinetochore function.