The methylation pattern of mouse mammary tumor virus (MMTV) proviral genes endogenous to the mouse strains C3H, 020, FM/JmsA, C57BL6, and BALB/c were investigated in various organs and mammary tumor tissue. Digestion of DNA with EcoRI or with EcoRI and HpaII followed by Southern blotting analysis and hybridization to a nick-translated MMTV DNA, allowed the distinction between the fully methylated and hypomethylated gene copies. MMTV proviral gene methylation was found to be organ specific, and the methylation pattern is stably inherited. The same proviral units present in different strains of mice exhibit the same organ-specific methylation patterns. Although proviral genes are normally heavily methylated in all tissues, hypomethylation of endogenous proviral genes was found in organs not known to express MMTV.