Mammalian centromere proteins (CENPs) can be divided into those that translocate from centromere to midzone in the progress of mitosis, and those that remain at the centromere throughout the cell cycle. The latter including CENP-A, CENP-B, and CENP-C is the candidate for DNA-binding protein. CENP-B has been shown previously to possess the specific DNA-binding activity to 17-base pair sequences dispersed on human centromeric alphoid repeats. In this study, we examined DNA-binding property of CENP-C that is localized to inner kinetochore plate of the metaphase chromosome. We independently isolated a full-length cDNA encoding human CENP-C and expressed it as the polypeptide tagged with histidine oligomer in Escherichia coli. After affinity purification with Ni(2+)-chelated resin, DNA-binding activity of the recombinant CENP-C renatured on the membrane was demonstrated by using human genomic DNA and an alphoid subfamily in South-Western-type blotting analysis. By constructing a series of truncated products, the DNA-binding domain was located at an internal 101-amino-acid stretch with no apparent homology to any other DNA-binding proteins. This may suggest that CENP-C is directly involved in formation of kinetochore chromatin fibers.