Mitochondrial oxygen tension (mitoPO(2)) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO(2) in vivo exists. Here we report in vivo measurement of mitoPO(2) and the recovery of mitoPO(2) histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO(2) in rat liver in vivo. The results demonstrate mitoPO(2) values of approximately 30-40 mmHg. mitoPO(2) was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO(2) distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.