It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.