The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 in-tracellular domain (Notch1^intra), deleted of most extracellular sequences, is overexpressed. Although Notch1^intra transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1^intra Tg mice that overexpress murine Notch1^intra in the mammary glands. We observed that MMTV/Notch1^intra Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of β-casein. Notch1^intra repressed expression of the β-casein gene promoter, as assessed in vitro with a β-casein/luciferase reporter construct. The MMTV/Notch1^intra Tg females developed mammary gland tumors, confirming the oncogenic po-tential of Notch1^intrain vivo. Furthermore, MMTV/Notch3^intra Tg mice exhibited a very similar pheno-type. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.