We present our strategy and progress towards the identification of rare restriction fragment length variants (RFLVs) segregating with the fragile‐X syndrome. DNA from a carrier mother and two retarded sons was digested with 7 restriction endonucleases and Southern blots were probed with cloned unique X‐chromosomal sequences. Two of 17 cloned segments tested revealed RFLVs between the X‐chromosomes of the carrier mother. One of them detected variants using
PvuII and MspI. The PvuII and MspI alleles found on the X‐chromosome bearing the fragile‐X mutation were not found in 31 and 22 random X‐chromosomes, respectively. The other probe detected variants using PvuII and TaqI. The TaqI allele present on the X‐chromosome with the fragile‐X mutation was found in 23 out of 25 random X‐chromosomes, while the PvuII allele was not found in 21 random X‐chromosomes. One of these probes and two other cloned unique X‐chromosome sequences were localized distal to Xq26 by in situhybridization to prometaphase chromosomes and by probing Southern blots containing DNA from a deleted X‐chromosome. These are being used for linkage analysis in an extended family.