The transplantation of primary mammary epithelial cells after adenovirus-Cre-mediated recombination provides a new approach for the study of specific gene function during mammary gland development and in breast cancer. Most mammary-gland-specific promoters identified to date are regulated by lactogenic hormones. They are expressed predominantly in lobuloalveolar cells during pregnancy and lactation, but not during early stages of ductal morphogenesis in the mammary epithelial cell progenitors, which are primarily implicated in tumorigenesis. In transgenic mice these promoters will continually or repeatedly express Cre depending on the hormonal environment precluding the definition of cell lineages. To circumvent these limitations, we have taken advantage of the unique regenerative capacity of mammary epithelium to reconstitute a mammary gland in an epithelium-cleared fat pad in conjunction with transient Cre expression using recombinant adenovirus in primary cultures. This approach was validated using mice carrying reporter constructs that exclusively express the LacZ gene after Cre-mediated deletion of a floxed DNA fragment. These studies demonstrated that, following recombination, cells that are marked as genetically manipulated contribute to the reconstitution of the mammary gland. The presence of beta-galactosidase-expressing cells in serial transplants of the primary outgrowths indicated that early progenitor or stem cells were successfully targeted. With the increased availability of floxed alleles, this approach should greatly facilitate the study of gene function during early stages of mammary gland development and in breast cancer.