The locus DXS98, detected with the 1.5-kb anonymous probe p4D-8, was recently shown to be closely linked and proximal to the locus for the fragile X syndrome, with theta = .05 at lod = 3.406, by utilizing a limited number of meioses informative for a two-allele MspI RFLP. Because DXS98 may be the closest available marker to the fragile X locus (FRAXA), we sought to increase its utility for linkage studies by extending its PIC and confirming its localization to Xq27, proximal to FRAXA. We have isolated 15 kb of genomic DNA (lambda 4D8-3) from the DXS98 locus by using p4D-8 to screen a genomic phage library containing partial Sau3A-digested human DNA. Three additional RFLPs for the enzymes BglII and XmnI were found by using the entire lambda 4D8-3 as probe. Combined heterozygosity for the four RFLPs in 25 unrelated females was 48%, as compared with only 28% when the MspI RFLP alone was used. In situ hybridization of unique sequences from lambda 4D8-3 was performed on metaphase chromosomes of lymphocytes and lymphoblasts from patients with the fragile X syndrome. Grains on the X chromosome were significantly clustered at band Xq27. Following fragile site induction, all nine grains in the q27-28 region were proximal to the fragile site. Confirmation of the location of DXS98 proximal to FRAXA and the new RFLPs at this locus make DXS98 more useful for linkage analysis and physical mapping in the region of the fragile X mutation.