The Xist gene responsible for X inactivation may take a unique chromatin structure because of exceptional expression from inactive X Chromosome, (Chr). We have examined differential chromatin packaging of the Xist gene region between active and inactive X Chr with a novel method consisting of the chromatin fractionation and allele-specific detection. Analysis of F1 heterozygous female mice from T(X;16)16H x MSM crosses and two cell clones derived from inter-subspecific F1 female mice demonstrated that the packaging level of the transcribed Xist region on inactive X Chr was as tight as that of the repressed Pgk-1 allele on the same chromosome. On the other hand, restriction endonuclease sensitivity assay of chromatin showed that the promoter region, but not transcribed regions, of the transcribed Xist allele retained accessibility to nucleases. These results may suggest a cis-element(s) in a regulatory region of the Xist gene to prevent the transcriptionally inhibitory effect of the chromatin packaging.