We have devised and tested a new strategy for selectively delivering molecules to tumor cells. Cellular association of polyarginine-based, cell-penetrating peptides (CPPs) is effectively blocked when they are fused to an inhibitory domain made up of negatively charged residues. We call these fusions activatable CPPs (ACPPs) because cleavage of the linker between the polycationic and polyanionic domains, typically by a protease, releases the CPP portion and its attached cargo to bind to and enter cells. Association with cultured cells typically increases 10-fold or more upon linker cleavage. In mice xenografted with human tumor cells secreting matrix metalloproteinases 2 and 9, ACPPs bearing a far-red-fluorescent cargo show in vivo contrast ratios of 2-3 and a 3.1-fold increase in standard uptake value for tumors relative to contralateral normal tissue or control peptides with scrambled linkers. Ex vivo slices of freshly resected human squamous cell carcinomas give similar or better contrast ratios. Because CPPs are known to import a wide variety of nonoptical contrast and therapeutic agents, ACPPs offer a general strategy toward imaging and treating disease processes associated with linker-cleaving activities such as extracellular proteases.