Preferential inactivation of the paternally derived X chromosome in extraembryonic membranes of female rodents has been clearly demonstrated, but the mode of X-chromosome inactivation in the human placenta has not been so clearly defined. We examined A and B variants of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in 42 informative placentae to investigate whether the earliest differentiating human female embryonic cells exhibit preferential inactivation of the paternally inherited X. Contamination of villi with fetal blood was eliminated through culture of villous tissues, and maternal cell contamination was eliminated by careful dissection. Both fresh and cultured amnion and chorion, as well as cultured villi, revealed preferential maternal allele expression.