Nitroreductase (NTR) activities have been known for decades, studied extensively in bacteria and also in systems as diverse as yeast, trypanosomes, and hypoxic tumors. The putative bacterial origin of mitochondria prompted us to explore the possible existence of NTR activity within this organelle and to probe its behavior in a cellular context. Presently, by using a profluorescent near-infrared (NIR) dye, we characterize the nature of NTR activity localized in mammalian cell mitochondria. Further, we demonstrate that this mitochondrially localized enzymatic activity can be exploited both for selective NIR imaging of mitochondria and for mitochondrial targeting by activating a mitochondrial poison specifically within that organelle. This constitutes a new mechanism for mitochondrial imaging and targeting. These findings represent the first use of mitochondrial enzyme activity to unmask agents for mitochondrial fluorescent imaging and therapy, which may prove to be more broadly applicable.