Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27. 3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X slte. A gene (FMR-1) was identified within a four cosmld contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRl genomic fragment, containing FMR-1 exonlc sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragife X chromosomes. This fragment contains a lengthy COG repeat that is 256 bp distal of the CpG island and maps within a FM?-1 exon. Localization of the brain-expressed FM?-7 gene to this EcoRl fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.