We have analyzed X-chromosome inactivation patterns in lymphocytes of 264 females from 38 families not known to have any genetic disease. Quantitative measures of X-inactivation showed strong sister–sister correlation in the degree of departure from equal numbers of cells having each X chromosome active, suggesting heritability of this phenotype. Strong sister–sister correlation was also observed for the fraction of cells having the same parent's X chromosome active, consistent with the possibility that this trait might be controlled by a cis-acting, X-linked gene. We used a sib-pair approach to determine whether X-inactivation phenotype was linked to loci in any region of the X chromosome. Both quantitative and discrete measures of X-inactivation phenotype showed evidence of linkage to markers in the region of the X inactivation center (XIC). The quantitative measure of X-inactivation phenotype used in our study also showed linkage to loci at Xq25–q26. This study provides the first evidence for X-linked inheritance of X chromosome inactivation phenotype derived from linkage analysis in phenotypically normal human families.