Trinucleotide repeat expansions have been implicated in the causation of a number of neurodegenerative disorders. In the case of fragile X syndrome, full expansion of the FMR1 repeat element (CGG)n has also been correlated with replication timing delay of the locus and proximal flanking sequences in male lymphoblasts. To define more extensively this altered region of DNA replication, as well as to extend these studies to female cells containing premutant and mutant alleles, study of the replication timing properties of a >2-Mb zone in the FRAXA region (Xq27.3-q28) was undertaken by using a FISH technique. In this assay, relative times of replication of specific loci are inferred from the ratios of singlet and doublet hybridization signals in interphase nuclei. In all individuals with a full expansion of the trinucleotide repeat, a large (1-1.2-Mb) region of delayed timing was observed; the apparent timing of the earlier-replicating allele in female cells in this region was intermediate between normal and affected alleles in males, which is in accordance with expectations of a mixed population of cells resulting from random X inactivation. In addition, expansion of the nearby FRAXE locus also was found to correlate with replication timing delay, although the extent of the altered region was somewhat less. Trinucleotide repeat expansion thus may be acting in the Xq27.3-q28 region to alter long-range chromatin structure that could influence transcription of gene sequences within the affected domain.