Specific chromosome domains in interphase nuclei of neurons and glia were studied by three-dimensional (3-D) reconstruction of serial optical sections from in situ hybridized human CNS tissue. Overall patterns of centromere organization, delineated with alphoid repeats, were comparable to those seen in mouse, and are clearly conserved in mammalian evolution. Cloned probes from other individual chromosome domains were used to define interphase organization more precisely. Homologous chromosomes were spatially separated in nuclei. In large neurons, probes specific for 9q12, or 1q12 showed that at least one homolog was always compartmentalized together with centromeres on the nucleolus, while the second signal either abutted the nucleolus or was on the nuclear membrane. A telomeric Yq12 sequence also localized together with perinucleolar centromeres in a completely non-Rabl orientation. In astrocytes, these three chromosome regions were on the membrane and not necessarily associated with nucleoli. Therefore there are different patterns of interphase chromosome organization in functionally distinct cell types. In contrast to the above domains, a 1p36.3 telomeric sequence embedded in a large Alu-rich and early replicating chromosome region, was always found in an interior euchromatic nuclear compartment in both neurons and glial cells. In double hybridizations with 1q12 and 1p36.3 probes, 1p arms were clearly separated in all cells, and arms projected radially into the interior nucleoplasm with non-Rabl orientations. There was no absolute or rigid position for each 1p arm with respect to each other or to the major dendrite, indicating that individual chromosome arms may be dynamically positioned even in highly differentiated cell types. We suggest that centromeric and other highly repeated non-transcribed sequence domains may act as general organizing centers for cell type specific interphase patterns that are conserved in mammalian evolution. Such centers would allow selected groups of chromosome arms to extend into (and contract from) an interior, presumably transcriptionally active, nuclear compartment.