CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro,the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo;and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.