To understand the molecular mechanisms by which the endogenous murine mammary tumor virus (MuMTV) proviruses are expressed and produce late-occurring mammary tumors in C3Hf mice, we analyzed, by the use of restriction enzymes and the Southern transfer procedure, genomic DNA from normal organs of mammary tumor-bearing and tumor-free mice and from 12 late-occurring C3Hf mammary tumors. We found, by using the restriction enzymes EcoRI and HindIII, that in addition to the preexisting endogenous MuMTV proviruses, new MuMTV-specific proviral DNA was integrated into new sites in the host genome in all 12 of the tumors that we examined. PstI digests of C3Hf tumor DNA revealed that the new proviral DNA found in C3Hf tumors was of endogenous origin. Moreover, the respective sizes of at least one of the new DNA fragments generated by EcoRI or HindIII digestion were the same in at least 50% of the C3Hf tumors analyzed, suggesting that the integration site of this new proviral DNA could be at the same location in the host genome of these tumors. Our results may imply that mammary tumorigenesis in C3Hf mice results from activation of cellular oncogenes by an MuMTV proviral DNA promoter. Specific hypomethylation of MuMTV proviral DNA was detected in the mammary tumors and spleens of C3Hf tumor-bearing mice. Our results indicated that most, if not all, of the hypomethylated MuMTV proviral DNA sequences were derived from the endogenous MuMTV provirus located at the MTV-1 locus, a locus responsible for the production of MuMTV antigens and increased incidence of mammary carcinoma in C3Hf mice. In spleens of non-tumor-bearing mice of ages 3, 6, 9, and 12 months, there was progressive hypomethylation of proviral DNA with increasing age, suggesting a possible correlation between demethylation of MuMTV proviral DNA in the spleens of C3Hf mice and the expression of endogenous MuMTV.