Abstract Fragile X syndrome, the most common inherited form of mental retardation syndrome, is caused by an expansion of the CGG trinucleotide repeat in the 5' UTR of the FMR1 gene, with concurrent hypermethylation of the region, which represses FMR1 expression. The syndrome is associated with the folate-sensitive chromosomal fragile site at Xq27.3 (FRAXA), where the gene responsible for the syndrome was first localized by linkage analysis using RFLP markers. In this study the linkage relationships of the RFLP markersat Xq27-28 and the characteristics of the CGG repeat expansion were investigated in northern Finnish fragile X families and molecular diagnostic methods were applied in order to improve diagnosis of the syndrome. Furthermore, the origin of fragile X mutations in the northern part of Finland was studied by haplotype analysis. Linkage studies were performed in 34 northern Finnish fragile X families/pedigrees using a total of 15 RFLPs (defining 11 loci). A refined genetic map around FRAXA including five RFLP markers having recombination fractions of 0.04 or less with FRAXA was obtained in an international study of 112 affected families, containing linkage data on twelve northern Finnish families. Linkage analysis significantly improved carrier detection in fragile X families compared with previous cytogenetic methods used in diagnosis. The most efficient RFLP-based protocol for carrier detection was proposed, which is based on use of the most adjacent markers and a minimum number of restriction enzymes. CGG repeat expansion of the FMR1 gene was investigated in original families collected for linkage studies and additional new ones. Large CGG repeat expansions (Δ > 500 bp) with concomitant methylation of the adjacent CpG island, i.e. full mutations, were found to be associated with mental retardation completely in males, but only 50% of the females having a full mutation were mentally impaired. Premutations (Δ < 700 bp) were found in healthy carriers. There was a size range of Δ = 500 to 700 bp, where the expansions could be either abnormally methylated or non-methylated, and it appeared that methylation is more important in determining the phenotype than the exact size of an expansion. Instability of the enlarged CGG repeats was detected, leading preferentially to size increases in successive generations. The instability of premutations was found to be stronger and the size increases larger in maternal than in paternal transmissions, and transition to a full mutation occurred only in female transmissions. In addition, the size of a maternal premutation was shown to have an important influence on the risk of its transition to a full mutation when transmitted. The critical premutation size leading invariably to full mutation in the offspring was found to be between Δ = 175 to 200 bp. In one of the studied families a rare contraction of a paternal premutation to a normal CGG repeat number in one of the daughters and further in her son was detected. Direct mutation analysis including measurement of the CGG repeat size and hypermethylation allowed unambiguous diagnosis of carriers and affected individuals in most cases. Haplotype analysis using two tightly linked microsatellite markers flanking the CGG repeat mutation was performed in 60 unrelated northern and eastern Finnish fragile X families. A significant difference was found in allelic and haplotypic distributions between normal X and fragile X chromosomes. A single haplotype, which was present only in 8% of the normal X chromosomes, accounted for 80% of the fragile X chromosomes. This enrichment of one fra(X) mutation in the Finnish population suggests founder effect.