The Rett syndrome (RS) is a degenerative neurological disorder occurring exclusively in young females. The disorder is sporadic in the majority of the cases, however a few familial cases with inheritance through maternal lines have been identified. Based on these observations the condition could be due to an X chromosome mutation which is lethal in males. To explain the familial cases, a hypothesis of possible non-random X inactivation is proposed. To investigate the possibility of non-random X chromosome inactivation in RS, we carried out analysis using restriction fragment length polymorphisms (RFLPs) and methylation sensitive enzymes at the PGK and HPRT loci. The results show that there is increased incidence of non-random X chromosome inactivation in peripheral blood leukocytes in sporadic RS patient (36%), as compared to healthy controls (8%). Using brain tissue from three patients, only a random pattern was detected, although varying degrees of skewing were detected in the peripheral tissues of these patients. Analysis of leukocyte DNA from a mother of two affected half-sisters revealed non-random X chromsome inactivation suggesting a possible selection against RS allele. Additional familial cases of RS should be evaluated to determine if this observation is common to all female carriers. If non-random X chromosome inactivation occurs in all the putative “carriers,” this would be the first evidence to support the hypothesis of an X linked mutation which is lethal in males. © 1990, All rights reserved.