Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.