Rett syndrome is a neurologic disorder characterized by early normal development followed by regression, acquired deceleration of head growth, autism, ataxia, and stereotypic hand movements. The exclusive occurrence of the syndrome in females and the occurrence of a few familial cases with inheritance through maternal lines suggest that this disorder is most likely secondary to a mutation on the X chromosome. To address this hypothesis and to identify candidate regions for the Rett syndrome gene locus, genotypic analysis was performed in two families with maternally related affected half-sisters by using 63 DNA markers from the X chromosome. Maternal and paternal X chromosomes from the affected sisters were separated in somatic cell hybrids and were examined for concordance/discordance of maternal alleles at the tested loci. Thirty-six markers were informative in at least one of the two families, and 25 markers were informative in both families. Twenty loci were excluded as candidates for the Rett syndrome gene, on the basis of discordance for maternal alleles in the half-sisters. Nineteen of the loci studied were chosen for multipoint linkage analysis because they have been previously genetically mapped using a large number of meioses from reference families. Using the exclusion criterion of a lod score less than -2, we were able to exclude the region between the Duchenne muscular dystrophy locus and the DXS456 locus. This region extends from Xp21.2 to Xq21-q23. The use of the multipoint linkage analysis approach outlined in this study should allow the exclusion of additional regions of the X chromosome as new markers are analyzed. This in turn will result in a defined region of the X chromosome that should be searched for candidate sequences for the Rett syndrome gene in both familial and sporadic cases.