We have constructed a 3.6 Mb sequence tagged sites (STS)-based yeast artificial chromosome (YAC) contig, consisting of 58 individual YAC clones, spanning the region PDHA1 and DXS451 on Xp22.1. In addition to establishing the order of PDHA1, ISPK-1, DXS2504, DXS1528 and the 13 known polymorphic loci as Xpter — PDHA1 — DXS443 — DXS3424 — ISPK-1 — DXS1229 — DXS2504 — DXS1528 — DXS365 — DXS7101 — DXS1683 — DXS1052 — DXS274 — DXS92 — DXS1226 — DXS41 — DXS989 — DXS451 — Xcen, we have also developed 35 novel STSs from YAC end clones. These results provide a high density of STS markers (approximately 1 per 70 kb). Furthermore, a detailed long-range restriction map of the contig has been constructed with rare-cutter enzymes and this has refined and verified the physical distances between markers inferred from YAC sizes and their STS content. The integration of the physical mapping data with previous genetic mapping data and the use of STSs and non-chimeric YAC clones reported here should facilitate the construction of a transcript map of this region and the positional cloning of disease genes in this portion of Xp22.1.