We recently showed that retroviral vectors can be targeted through protease substrate interactions. Infectivity is blocked by a polypeptide fused to the viral envelope glycoprotein (SU) and is restored when a protease cleaves the connecting linker, releasing the inhibitory polypeptide from the viral surface. Protease specificity is achieved by engineering the sequence of the linker. Here, using two different matrix-metalloproteinase (MMP)-activatable vectors, we demonstrated highly efficient and selective transduction of MMP-rich target cells in a heterogeneous cell population. In vivo, the MMP-targeted vectors showed strong selectivity for MMP-rich tumor xenografts. Protease-activatable vectors offer new possibilities for in vivo targeting of gene delivery.