A yeast artificial chromosome (YAC) contig of approximately 2.3 Mb has been constructed for the region of the mouse X chromosome between Gabra3 and G6pd. YACs were isolated from four genomic libraries by PCR or hybridization screening for the loci Gabra3, DXHX1104, F8a, DXHX52, DXBay2, L1cam, and Rsvp. Orientation of YACs was performed by isolation of ends using several PCR-based methods, including an adaptation for mouse YAC analysis of interspersed repetitive sequence (IRS)-vector PCR. YAC ends were mapped on a panel of somatic cell hybrids, in an interspecific backcross, using pulsed-field gel (PFG) electrophoresis or by hybridization to other YACs in the contig. Overlapping YACs were further aligned by restriction mapping using rare-cutter restriction endonucleases, as well as by comparison of IRS-PCR fingerprints. This contig represents the largest one assembled to date in the mouse. It should facilitate the isolation of genes in the region and may aid in the completion of a physical contig for the homologous human region, Xq28.