The patterns of the milk-transmitted (exogenous) mouse mammary tumor virus (MuMTV) DNA restriction endonuclease fragments in the nodule and tumor stages of BALB/cfC3H mouse mammary neoplasia were compared with the use of the Southern blot analysis. Acquired MuMTV restriction fragments were detected in DNA from hyperplastic alveolar nodules (HAN), from primary hyperplastic outgrowths (HPO), from families of transplanted HPO, from tumors from HPO, and from serially transplanted tumors. The restriction fragment patterns suggested that the HAN were composed of clonal dominant populations. Transplantation of subdivisions of individual HAN resulted in HPO with DNA restriction patterns suggesting that HAN also contained two or more subpopulations. In all cases, HAN subpopulations shared MuMTV restriction fragments suggesting a common origin. Forty-seven tumors arising from HPO shared MuMTV restriction fragments with the HPO. Most but not all tumors had additional acquired MuMTV restriction fragments not detected in the progenitor HPO, indicating that they were composed of a distinct subpopulation that originated from the HPO. The restriction fragment pattern in some tumor lines was remarkably stable through many transplant generations. Some tumors had no major additional restriction fragments, suggesting that major rearrangements of MuMTV DNA are not required for tumorigenesis.