We have screened for the presence of two centromere autoantigens, CENP-B (80 kDa) and CENP-C (140 kDa) at the inactive centromere of a naturally occurring stable dicentric chromosome using specific antibodies that do not cross-react with any other chromosomal proteins. In order to discriminate between the active and inactive centromeres on this chromosome we have developed a modification of the standard methanol/acetic acid fixation procedure that allows us to obtain high-quality cytological spreads that retain antigenicity with the anti-centromere antibodies. We have noted three differences in the immunostaining patterns with specific anti-CENP-B and CENP-C antibodies. (1) The amount of detectable CENP-B varies from chromosome to chromosome. The amount of CENP-C appears to be more or less the same on all chromosomes. (2) CENP-B is present at both active and inactive centromeres of stable dicentric autosomes. CENP-C is not detectable at the inactive centromeres. (3) While immunofluorescence with anti-CENP-C antibodies typically gives two discrete spots, staining with anti-CENP-B often appears as a single bright bar connecting both sister centromeres. This suggests that while CENP-C may be confined to the outer centromere in the kinetochore region, CENP-B may be distributed throughout the entire centromere. Our data suggest that CENP-C is likely to be a component of some invariant chromosomal substructure, such as the kinetochore. CENP-B may be involved in some other aspect of centromere function, such as chromosome movement or DNA packaging.