To investigate X-chromosome inactivation (XCI) in human trophoblasts during early pregnancy, trophoblast genomic DNA was extracted and analyzed for a Bst XI restriction endonuclease site polymorphism in the X-linked phosphoglycerate kinase gene, after digestion with methylation-sensitive Hpa II (control samples were digested instead with Afa I). Six villous trophoblast DNA samples were informative for the polymorphism (ie, heterozygous) and were derived from women homozygous for the polymorphism. These samples were then evaluated for XCI. In five of the six samples with Hpa II predigestion, the sizes of the two heterozygous band peaks differed; maternal X-chromosome (X(M))-derived alleles showed smaller peak sizes than paternal X-chromosome (X(P))-derived alleles, but the differences varied in degree. In samples obtained by microdissection from formalin-fixed, paraffin-embedded tissues (30 samples from different anchoring villi, and 38 samples from different branch villi), monoclonal band patterns of X(P)-derived alleles were observed more frequently than those of X(M)-derived alleles, but almost half of the samples showed polyclonal patterns. Our results suggest that a skewing of XCI exists in the human trophoblast; however, the degree of nonrandomness due to predominant X(P) inactivation appears to be restricted. It is probable that transcription of the X inactivation center (XIC) begins earlier in mice than in humans.