Fluorescence in situ hybridization and multispectral confocal laser scanning microscopy revealed a highly dynamic nucleolar-associated chromosome 15 satellite III heterochromatin cluster in humans. This nucleolar-associated DNA was highly decondensed at the metaphase plate compared with its topography at interphase and appeared to act as a centre for the post-mitotic reorganization of the nucleolus. Our data showed unexpected trans-mitotic changes in the topography of this nucleolar-associated satellite III DNA that suggest that this locus-specific heterochromatin superstructure may be involved in nucleolar organization.