The recent identification of an abnormally amplified trinucleotide (cytosine guanine guanine) repeat in the fragile X gene (FMR-1) of males with fragile X syndrome and their carrier mothers allows the study of the mutation in individuals at risk. In this report, data on 396 patients and 35 normal controls are reported. Included in this sample are patients with no known family history of fragile X syndrome or mental retardation for whom the risks of fragile X syndrome are unclear. All 39 cytogenetically positive affected males and six females had the full mutation, as represented by a restriction fragment size increase (delta) of 500 base pairs (bp) or more within the cytosine guanine guanine repeat-bearing fragment of the FMR-1 gene; and all 16 of the normal obligate carrier females bore the premutation, as demonstrated by a delta of 100 to 500 bp. Of 124 patients (62 males and 62 females) with a family history of fragile X syndrome, five (8%) of the males and 25 (40%) of the females had the premutation. Five (2.2%) of the 231 mentally impaired patients with no confirmed family history of fragile X syndrome were found to have the full mutation. Twelve (33%) of 36 mentally impaired males and one (20%) of five females with unknown family history were found to carry an amplified cytosine guanine guanine repeat. Using this technique, we also reevaluated risk assessments previously generated by linkage analysis and unambiguously determined the carrier status of individual family members.