Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. However, the underlying cellular/molecular mechanisms remain unknown. Amplification of ErbB2, a receptor tyrosine kinase, is found in 20-30% of breast cancer patients. Ethanol preferably stimulates invasion by breast cancer cells over-expressing ErbB2 in vitro. Over-expression of ErbB2 is positively associated with elevated levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. Ethanol at physiologically relevant concentrations activates MMP-2 without altering its expression level in mammary epithelial cells over-expressing ErbB2, but not in cells expressing low levels of ErbB2. The activation is dependent on c-jun N-terminal kinases (JNK) and reactive oxygen species. Selective inhibitors of MMP-2 and anti-oxidants significantly inhibit ethanol-stimulated cell invasion. Similarly, knocking down MMP-2 by small interference RNA induces a partial blockage on ethanol-promoted cell invasion. Matrix metalloproteinase-2 is predominantly expressed in stromal fibroblasts; ethanol also activates fibroblastic MMP-2. The conditioned medium collected from ethanol-exposed fibroblasts dramatically stimulates the invasion of breast cancer cells. The role of MMP-2 in ethanol-induced tumor promotion is discussed.