The XIST gene plays an essential role in X Chromosome (Chr) inactivation during the early development of female humans. It is believed that the XIST gene, not encoding a protein, functions as an RNA. The XIST cDNA is unusually long, as its full length is reported to be 16.5 kilobase pairs (kb). Here, comparison of sequences from the genomic interval downstream to the 3' end of the human XIST gene against the human EST database brought to light a number of human EST sequences that are mapped to the region. Furthermore, PCR amplification of human cDNA libraries and RNA fluorescence in situ hybridization (RNA-FISH) demonstrate that the human XIST gene has additional 2.8 kb downstream sequences which have not been documented as a part of the gene. These data show that the full-length XIST cDNA is, in fact, 19.3 kb, not 16.5 kb as previously reported. The newly defined region contains an intron that may be alternatively spliced and seven polyadenylation signal sequences. Sequences in the newly defined region show overall sequence similarity with the 3' terminal region of mouse Xist, and three subregions exhibit quite high sequence conservation. Interestingly, the new intron spans the first two sub-regions that are absent in one of the two isoforms of mouse Xist. Taken together, we revise the structure of human XIST cDNA and compare cDNA structures between human and mouse XIST/Xist. al. 1992). This gene, called XIST/Xist (X inactive specific transcript), shows several interesting features. First, both human and mouse XIST/Xist cDNA are unusually long, reportedly 16.5 kb and 17.8 kb, respectively (Brown et al. 1992; Hong et al. 1999). Second, the transcript does not seem to encode a protein, on the basis of the lack of a significant open reading frame, absence of the Xist RNA from polysomes, and localization of the transcript in the nucleus (Brockdorff et al. 1992; Brown et al. 1992). Third, the XIST/Xist RNA physically associates with, or 'coats,' the inactive X Chr (Brown et al. 1992; Clemson et al. 1996). Fourth, XIST/Xist transcripts can be observed as early as the four-cell stage, and upon the initiation of X-inactivation, the steady-state level of the transcript rises dramatically, apparently by stabilization of the RNA (Panning et al. 1997; Sheardown et al. 1997). Although the function of XIST/Xist is not known, deletion of the gene leads to failure of X-inactivation, and knock-out mice die around the gastrulation stage (Marahrens et al. 1997; Penny et al. 1996). In this report, we revise the structure of the human XIST cDNA and discuss structural features of the newly defined region.