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Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGKI). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.Female primordial germ cells of mammals resemble somatic cells in having both an active and an inactive X chromosome. Evidence for inactivity of one X chromosome of oogonia has been found both cytologically and by studies on glucose-6-phosphate dehydrogenase heterodimer formation (1, 2). After migration of the oogonia to the genital ridge at, or shortly before, the time of entry into meiosis, reactivation of the previously inactive X chromosome occurs (1-6). DNA methylation has been correlated with X chromosome inactivation in many studies (reviewed in refs. 7-9) and it is of interest to determine whether a loss of methylation is concomitant with X chromosome reactivation during oogenesis. Data on DNA methylation of oogenic cells are sparse. Several years ago evidence was obtained for undermethylation of some repeated sequences in female germ-line DNA (10, 11), and more recently, Driscoll and Migeon (12) obtained evidence for lack of methylation of several X-linked and autosomal genes in oogenic and spermatogenic cell fractions.As the latter study was done on tissue fractions rather than preparations free of somatic cells, we thought it was important to measure DNA methylation of highly purified germ cells. As such cells are isc. table only in small quantities, we used a polymerase chain raction (PCR)-based assay previously used to measure DNA methylation at a CCGG site in individual mouse embryos (13).The site we chose to assay was Hpa II site H8 in the 5' untranslated coding region of the human X-linked PGKI gene, 20 base pairs (bp) downstream of the major transcription start site (14). The 5' region of the PGKI gene is now the most highly characterized X-linked promoter region, and the relationship between methylation, transcription, and transcription factors has been studied both in normal human lymphocytes and in cultured cell lines (15)(16)(17)(18)(19). Recently, ligation-mediated genomic sequencing has been used to establish that the promoter region of the PGKI gene on the inactive X chromosome is methylated at 60 of 61 CpG sites, whereas the active promoter has no methylated sites (19). Prior to these studies, methylation-sensitive restriction enzymes had been used to investigate methylation at the 8 Hpa II sites in the PGKI 5' region in lymphocytes (15) and in cell lines before and after...