Activatable cell-penetrating peptides (ACPPs) are a new class of promising molecular imaging probes for the visualization of enzymes in vivo. The cell-penetrating function of a polycationic peptide is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a cleavable linker present between the polycationic cell-penetrating peptide and polyanionic peptide affords dissociation of both domains and enables the activated cell-penetrating peptide to enter cells. Here, we aimed to develop an ACPP sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for nuclear imaging purposes. Methods: MMP-2/9 ACPPs and nonactivatable cell-penetrating peptides (non-ACPP) were prepared by 9- fluorenylmethyloxycarbonyl solid-phase peptide synthesis and labeled with 177Lu or 177Lu/125I for dual-isotope studies. The in vivo biodistribution of these probes was assessed in MMP-2/9– positive tumor-bearing mice (n 5 6) and healthy mice (n 5 4) using g-counting. Furthermore, a radiolabeled cell-penetrating peptide serving as a positive control was evaluated in tumorbearing mice (n 5 6). Results: Biodistribution studies showed a5-fold-higher retention of ACPP in tumor than in muscle (P , 0.01) and a 6-fold-higher tumor retention relative to non-ACPP (P , 0.01), supporting earlier studies on fluorescently labeled ACPPs proposing activation by tumor-associated MMP-2/9. Surprisingly, however, the uptake of ACPP was significantly higher than that of non-ACPP in almost all tissues (P , 0.01). To unravel the activation process of ACPP in vivo, we developed dual-isotope ACPP analogs (dACPPs) that allowed us to discriminate between uncleaved dACPP and activated dACPP. In vivo biodistribution of dACPP indicated that the tissue-associated counts originated from activated dACPP. Interestingly dACPPadministration to healthy mice, compared with MMP- 2/9–positive tumor-bearing mice, resulted in a similar dACPP biodistribution. Furthermore, a radiolabeled cell-penetrating peptide showed tumor-to-tissue ratios equal to those found for ACPP (P . 0.05). Conclusion: This study demonstrates that the tumor targeting of radiolabeled MMP-2/9 ACPPs is most likely caused by the activation in the vascular compartment rather than tumor-specific activation, as suggested earlier. The results in the present paper indicate that different and more tissue-specific enzyme–ACPP combinations are needed to unleash the full potential of the elegant ACPP concept in living animals.