Cancer stem cells, initiating and sustaining the tumor process, have been isolated in human and murine breast cancer using different cell markers. In the present study, we aimed to evaluate the presence and characteristics of stem/tumor-initiating cells in the model of the mouse mammary neoplasia driven by the activated form of rat Her-2/neu oncogene (BALB-neuT mice). For this purpose, we generated tumor spheres from primary spontaneous BALB-neuT tumors. Tumor sphere cultures were characterized for clonogenicity, self-renewal, and ability to differentiate in epithelial/myoepithelial cells of the mammary gland expressing basal and luminal cytokeratins and alpha-smooth muscle actin. In addition, tumor spheres were more resistant to doxorubicin compared with parental tumor cells. In the attempt to identify a selected marker for the sphere-generating cells, we found that Sca-1(+) cells, present in tumors or enriched in mammospheres, and not CD24(+) or CD29(+) cells, were responsible for the sphere generation in vitro. Moreover, cells from the tumor spheres showed an increased tumor-generating ability in respect to the epithelial tumor cells. Sca-1(+) sorted cells or clonal mammospheres derived from a Sca-1(+) cell showed a superimposable tumor-initiating ability. The data of the present study indicate that a Sca-1(+) population derived from mammary BALB-neuT tumors is responsible for sphere generation in culture and for initiating tumors in vivo.