We have constructed a detailed physical map of the 35 Mb region spanning human chromosome Xp22.3-Xp21.3. The backbone of the map is represented by a single oriented contiguous stretch of 585 overlapping yeast artificial chromosome (YAC) clones covering the entire region. The map is formatted with 615 map objects that include 324 YACs, 185 sequence tagged sites, 28 genes, 85 chromosomal breakpoints and 37 highly polymorphic markers. Physical mapping was both guided and confirmed using 183 bins defined by chromosomal breakpoints and by overlapping regions of YAC clones. The localization of polymorphic markers in the physical map permits the integration of physical and genetic data across the region. These data establish chromosome Xp22.3-Xp21.3 as one of the best characterized large regions in the human genome. The map should greatly facilitate finer scale mapping and sequencing as well as the identification of disease genes from this portion of the human genome.