Part of the higher-order structure of chromatin is achieved by constraining DNA in loops ranging in size from 30 to 100 kilobase pairs; these loops have been implicated in defining functional domains and replicons and possibly in facilitating transcription. Because the human active and inactive X chromosomes differ in transcriptional activity and replication, we looked for differences in their chromatin loop structures. Since the islands of CpG-rich DNA at the 5' ends of X-linked housekeeping genes are the regions where functional differences in DNA methylation and nuclease sensitivity are found, we looked for scaffold association of these sequences after extraction of histones with lithium diiodosalicylate. Specifically, we examined the 5' CpG islands within the hypoxanthine phosphoribosyltransferase, glucose 6-phosphate dehydrogenase, P3, GdX, phosphoglycerate kinase type 1, and a-galactosidase loci in human lymphoblasts obtained from individuals with 1 to 4 X chromosomes. Although we detected no scaffold-associated regions near these genes, we found several such regions at the ornithine transcarbamylase and blood clotting factor IX loci. Our results suggest that the CpG islands are excluded from the nuclear scaffold and that even though transcriptionally active, housekeeping genes are less likely than X-linked tissue-specific genes to be scaffold associated. In all cases, the pattern of scaffold association was the same for loci on active and inactive X chromosomes.Sex dosage compensation in mammalian females is achieved by random inactivation of one X chromosome (reviewed in reference 26). Active and inactive X chromosomes differ in a number of characteristics, including degree of condensation (3), timing of replication (18), and transcriptional activity (19). In addition, genes that are constitutively expressed are methylated differently on the two chromosomes (24,46,49,50). The functional differences in DNA methylation reside in the clusters of CpG dinucleotides, the so-called CpG islands, that are commonly found at the 5' ends of many housekeeping genes (4). These clusters are methylated only in genes on the inactive X chromosome, and methylation helps maintain the inactive state (23). Furthermore, since these clusters are nuclease hypersensitive in active but not in inactive genes (51), the chromatin structures of the two chromosomes must differ. Finally, methylation and gene activity are coordinately regulated at three CpG clusters near the glucose 6-phosphate dehydrogenase (G6PD) locus (46), which suggests that CpG islands may help define functional domains along the chromosome.Although the differences in structure of the homologous X chromosomes during interphase are striking, little is known about the organization of X chromatin at the molecular level. The 300-A (30-nm) chromatin fiber is thought to be organized into 30-to 100-kilobase-pair (kbp) loops that are anchored to a protein structure variously termed the nuclear matrix (7,8,37,(41)(42)(43)48), scaffold (9, 15-17, 21, 22, 25, 33, 34). or cage...