HER2/neu overexpression leads to poorer prognosis and higher risk of disease reoccurrence in breast cancer patients. The causative factors responsible for increasing HER2/neu expression levels on mammary cells are not known. We investigated whether factors associated with inflammation or metastasis could induce HER2/neu expression on human mammary epithelial cells (HMECs). Human mammary epithelial cells and several human breast cancer cell lines used in our studies were treated with several agents, including estrogen and matrix metalloproteinase-9 (MMP-9), either alone or in various combinations. Relative expression of HER2/neu on the surface of target cells was assessed using fluorochrome-tagged antibodies and a fluorescence cytometer. HER2/neu gene expression was also determined by Western blot analysis and PCR. Apoptosis levels were also determined. MMP-9, administered either alone or in combination with interleukin-7 and estrogen, caused a significant rise in HER2/neu expression on the surface of HMECs. The induction in HER2/neu protein expression was suppressed using a MMP-9 inhibitor. Similar results were obtained for breast cancer cells treated with the estrogen in combination with MMP-9. MMP-9 treatment significantly decreased apoptotic levels in HMECs. Our results indicate that MMP-9 is a regulator of HER2/neu expression on human mammary epithelial cells and suggest that upregulation of HER2/neu by MMP-9 may be relevant to altering the characteristics of normal mammary cells toward a transformed phenotype.