Fragile X syndrome, the most common form of familial mental retardation, is mainly caused by the expansion of an unstable region of CGG repeats in the 5' untranslated region of the FMR1 (Fragile X Mental Retardation-1) gene. Molecular tools to detect an abnormal CGG expansion in FMR1 include Southern blot hybridization and PCR amplification. Southern blotting with the StB12.3 probe and Eco RI/Eag I double digestion is widely used as a routine test for fragile X syndrome diagnosis in laboratories around the world. A patient with mental retardation of unknown origin showed absence of digestion for Eag I due to a -149C-->G substitution in the CpG island of the FMR1 gene, which destroys that restriction enzyme site. Screening for other changes around that region also detected a -154insGGC in a patient with a phenotype highly suggestive of fragile X syndrome but without CGG expansion. Expression studies did not show any abnormal changes in FMR1 function. In summary, we have identified two different changes (a C to G substitution at -149 and a GGC insertion at -154) in the promoter of the FMR1 gene. These are the first variants described in the promoter of the FMR1 gene.