The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins-polycomb, polyhomeotic and posterior sex combs-in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression (Csink, A.K., and S. Henikoff. 1996. Nature. 381:529-531 ), there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. We determined that <2% of PH remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG-mediated gene repression.