CENP-G is a novel constitutive centromere-specific protein localized to the kinetochore inner plate and subjacent region. It has been identified as associating specifically with the alpha-1 subfamily of alpha-satellite DNA. In the present work, the localization of CENP-G was compared with that of other CENPs by immunofluorescence and fluorescence in situ hybridization. Studies were carried out on four abnormal human centromeres: two neocentromeres and two inactive centromeres. CENP-G was detected in one of the two inactive centromeres but not in the other that shows a partial deletion of the alphoid DNA. Interestingly, CENP-G is also present in neocentromeres, which lack alphoid DNA sequences, and in the human Y chromosome, which lacks the alpha-1 type of satellite DNA. These data provide further evidence that CENP-G may be an essential factor in centromeric function and that in centromeres lacking the alpha-1 subfamily of alphoid DNA, other DNA sequences are able to bind CENP-G.