The organization and expression of germinally transmitted mouse mammary tumor virus (MMTV) proviruses in C3Hf/HeSed mouse tissues were examined. Digestion with the restriction enzymes EcoRI, BamHI, and HindIII and hybridization with cloned probes specific for the long terminal repeat and the 5' and 3' regions of the MMTV genome revealed three full-length (units Ib, II, and V) and two subgenomic (units I and IX) MMTV proviruses in C3Hf/HeSed mouse germ line DNA. The EcoRI fragments (15.0 and 5.7 kilobase pairs [kbp]) that contained unit Ib were previously described as separate, subgenomic MMTV proviruses. The methylated state of each full-length MMTV provirus was examined in DNA from C3Hf/HeSed mouse livers, spleens, mammary glands, and mammary tumors by digestion with EcoRI or BamHI in combination with the methyl-sensitive restriction enzymes HhaI or HpaII. Unit Ib contained HhaI- and HpaII-sensitive sites in spleen, mammary gland, and mammary tumor DNA but was completely methylated in liver DNA. Units II and V contained HhaI- and HpaII-sensitive sites in mammary gland and mammary tumor DNA, but the sites were extensively methylated in spleen and liver DNA. The HhaI-sensitive sites were mapped to the 5' end of the 5' and 3' long terminal repeats of each full-length MMTV provirus. C3Hf/HeSed mouse tissue RNA was examined for MMTV transcripts. Mammary glands contained MMTV RNA species of 9.0, 3.8, and 1.7 kb. Mammary tumors contained high levels of the 9.0- and 3.8-kb transcripts but lacked the 1.7-kb species. A very low level of the 3.8-kb MMTV transcript was present in spleens. Livers lacked detectable MMTV RNA. These results implicate mammary tissue as the site of unit V activation in the formation of MMTV virions.