Molecular oxygen is the primary oxidant in biological systems. The ultimate destination of oxygen in vivo is the mitochondria where it is used in oxidative phosphorylation. The ability of this process to produce an amount of high-energy phosphates adequate to sustain life highly depends on the available amount of oxygen. Despite a vast array of techniques to measure oxygen, major (patho)physiological questions remain unanswered because of the unavailability of quantitative techniques to measure mitochondrial oxygen in situ. Here we demonstrate that mitochondrial PO(2) can be directly measured in living cells by harnessing the delayed fluorescence of endogenous protoporphyrin IX (PpIX), thereby providing a technique with the potential for a wide variety of applications. We applied this technique to different cell lines (V-79 Chinese hamster lung fibroblasts, HeLa cells and IMR 32-K1 neuroblastoma cells) and present the first direct measurements of the oxygen gradient between the mitochondria and the extracellular volume.