We have investigated whether repression of the putative tumor suppressor gene BARD1 or expression of the Notch4(int-3) oncogene in non-tumorigenic mammary epithelial cells affects their in vitro morphogenetic properties. Bard1 (Brca1-associated ring domain) is a protein interacting with Brca1 and thought to be involved in Brca1-mediated tumor suppression. To investigate the potential role of Bard1 in mammary gland development, we repressed its expression in TAC-2 cells, a murine mammary epithelial cell line which, when grown in three-dimensional collagen gels, forms branching ducts in response to hepatocyte growth factor (HGF) and alveolar-like cysts in response to hydrocortisone. Whereas Bard1 repression did not markedly modify the tubulogenic response of TAC-2 cells to HGF, it dramatically altered cyst development, resulting in the formation of compact cell aggregates devoid of central lumen. In addition, when grown to post-confluence in two-dimensional cultures, Bard1-suppressed TAC-2 cells overcame contact-inhibition of cell proliferation and formed multiple cell layers. The Notch4(int-3) oncogene, which codes for a constitutively activated form of the Notch4 receptor, has been reported to induce undifferentiated carcinomas when expressed in the mammary gland. The potential effect of activated Notch4 on mammary gland morphogenesis was investigated by retroviral expression of the oncogene in TAC-2 cells. Notch4(int-3) expression was found to significantly reduce HGF-induced tubulogenesis and to markedly inhibit hydrocortisone-induced cyst formation. In addition, Notch4(int-3) expressing TAC-2 cells formed multilayers in post-confluent cultures and exhibited an invasive behavior when grown on the surface of collagen gels. Taken together, these results indicate that both repression of Bard1 and expression of Notch4(int-3) disrupt cyst morphogenesis and induce an invasive phenotype in TAC-2 mammary epithelial cells.