Existing methodologies have been combined to produce a directed approach to the isolation of human genes that escape X inactivation. A mouse-human somatic cell hybrid line was established that has an inactive X as its only human chromosome, and nuclear RNA from this cell line was used to construct a cDNA library. Transcribed human sequences were isolated by screening the library with labeled human DNA. The corresponding genomic sequences were isolated in phage or cosmid clones, and exons were identified by detection of transcripts on northern blots. By these means three human loci have been identified that contain genes expressed from an inactive X chromosome. Fluorescence in situ hybridization has been used to map these genes to Xp21.1-22.1, Xp22.1-22.2, and terminal Xp/Yp. One of the three genes (XE45) corresponds to the ZFX gene, while the other two genes (XE7 and XE59) represent novel cloned sequences. Physical and genetic evidence indicate that XE7 is a newly identified pseudoautosomal gene.