The largest of the commonly used probes for Southern blot diagnosis of fragile X mental retardation syndrome spans the CGG repeat cluster in the FMR-1 gene. This probe causes the appearance of 'common' or 'constant' background bands which occasionally complicate the interpretation of autoradiographic results. By removing a 357 bp Sphl to Nhel fragment containing the CGGs from the probe pE5.1, we constructed a probe which eliminates the background bands yet allows the use of a large probe (4.8 kb) to detect changes in the diagnostic 5.2 kb genomic EcoRl band. This CGG-deficient probe has been used in routine diagnostic cases as well as in second round testing of pE5.1-probed cases where enlarged mutant bands are suspected to comigrate with the background bands.