The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb. Xi-territories were characterized by a rounder shape as compared to the flatter and more extended shape of Xa-territories. The overall compaction of the entire Xi-territory, including the Barr body, was only 1.2-fold higher than the Xa-territory. Significant differences, however, were noted between distinct subchromosomal segments: At 20 Mb length scales higher compaction in Xi-territories was restricted to specific segments, but higher compaction in these segments was not correlated with gene density, transcriptional activity, LINE content or histone markers locally enriched in Xi-territories. Notably, higher compaction in Xi-territories observed for 20 Mb segments was not reflected accordingly by inclosed segments of 1-4 Mb. We conclude that compaction differences result mainly from a regrouping of ~1 Mb chromatin domains rather than from an increased condensation of individual domains. In contrast to a previous report, genes subject to inactivation as well as escaping from inactivation were not excluded from the interior of the Barr body. © 2011 Landes Bioscience.