Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing “side population” properties. Sca-1^pos cells in the mammary gland were localized to the luminal epithelia by using Sca-1^+/GFP mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1^pos population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1^neg cells when transplanted into the cleared mammary fat pads of host mice.