We are developing protease-activatable gene delivery vehicles for selective gene delivery to protease-expressing cells. Angiogenesis, inflammation, and cancer invasion are linked to the overexpression of matrix metalloproteinases (MMPs), which destroy the extracellular matrix. Therefore, the MMPs are promising targets for therapy. We have displayed epidermal growth factor (EGF) on retroviral vector particles as an MMP-cleavable amino-terminal extension of the 4070A murine leukemia virus (MLV) envelope glycoprotein. This was achieved by engineering an MMP-cleavage signal (PLGLWA) into the linker between the EGF domain and the 4070A SU. The chimeric envelope was expressed and incorporated into viral particles, and the EGF domain could be cleaved from the surface of the viral particles by gelatinase A (MMP-2). The MMP-sensitive vector and control MMP-insensitive vectors could bind, via their displayed EGF domains, to EGF receptors on A431 cells but were unable to infect them because the EGF receptor (EGFR) does not support postbinding steps required for retroviral entry. In the presence of exogenous MMPs, the infectivity of the MMP-sensitive vector, but not of the MMP-insensitive vectors, was restored on A431 cells, and this cleavage activation could be partially blocked by MMP inhibitors. Endogenous MMPs produced by EGFR-positive HT 1080 cells could selectively activate the MMP-sensitive vector giving rise to a titer that was 1,000-fold higher on HT 1080 cells than on MMP-negative A431 cells. Inhibitor studies and gelatin zymograms indicated that the membrane-associated MT-MMP expressed on the HT 1080 cells played an important role in cleavage activation of the vector. When presented simultaneously with both EGFR-positive cell lines A431 and HT 1080, the vector could efficiently discriminate between the two different cell types, infecting the MMP-positive HT 1080 cells in preference over the A431 cells.