The process of cancer cell invasion involves degradation of the extracellular matrix (ECM) by proteases, integrin adhesion and cell motility. The role of ECM degrading proteases on the hypoxia-induced invasion of breast carcinoma cells was investigated. Hypoxia markedly increased the invasion capacity of MDA-MB-231 and MDA-MB-435 breast carcinoma cell lines. Matrix metalloproteinase (MMP) inhibitors blocked the hypoxia-induced invasion, whereas other protease inhibitors had no effect. Antibodies or siRNAs blocking either membrane type-1 MMP (MT1-MMP) or MMP-2 were effective in reducing the hypoxia-induced invasion. Serum-free reconstitution experiments confirmed the involvement of the MT1-MMP/MMP-2/tissue inhibitor of metalloproteinase-2 complex in this hypoxia-induced response. Overexpression of MT1-MMP in a poorly invasive breast cancer cell line, T47-D, promoted hypoxia-induced invasion and MMP-2 activation. Cell surface accumulation and activation of MT1-MMP without apparent regulation at the mRNA or protein levels indicated a post-translational adaptive response to hypoxia. Inhibition of the small GTPase RhoA eliminated the hypoxia-induced invasion and blocked the localization of MT1-MMP to the plasma membrane. Zymographic and molecular analysis of human breast tumors showed a strong correlation between hypoxic microenvironments and MMP-2 activation without changes in MT1-MMP expression. Our studies suggest that hypoxic tumor microenvironments promote breast cancer invasion through an MT1-MMP-dependent mechanism.