We have microdissected and cloned the region around the fragile site at Xq27.3 on the human X chromosome. All of the clones tested map to the Xq27-Xq28 region, and detailed mapping on a panel of somatic cell hybrids indicates that the microdissected library contains sequences derived from both sides of the fragile X mutation. Some of these clones give signals in rodent DNA. This library demonstrates the power of microdissection for the identification of potential coding sequences near a disease locus and provides a promising resource for the identification of the fragile X mutation.