Cosmids, isolated from a 475 kb YAC that spans the fragile X region, and the YAC itself, were used for fluorescence in situ hybridization (FISH) on metaphase chromosomes from fragile X patients. Cosmid 22.3, containing most of the hybrid translocation breakpoints, shows in situ hybridization signals distal and proximal from the fragile X site. We propose that the size of the fragile site is limited to 20 kb.