The protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) has been recently introduced as the first method to measure mitochondrial oxygen tension (mitoPO2) in living cells and tissues. The current implementation of the technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of 5-aminolevulinic-acid-enhanced mitochondrial PpIX. It represents a significant step forward in our ability to comprehensively measure tissue oxygenation. PpIX-TSLT is feasible for application in humans and recently we have been able to measure for the first time mitoPO2 in humans. MitoPO2 in intact tissues reflects the balance between oxygen supply and demand at the cellular level. Administration of aminolevulinic acid induces measurable mitochondrial levels of PpIX. PpIX acts as a mitochondrially located oxygen-sensitive dye by emitting a red delayed fluorescence after excitation with a pulse of green light. The lifetime of the delayed fluorescence is inversely related to PO2 by the Stern-Volmer equation. In vivo measurements of mitoPO2 in liver, heart, and skin of rats have revealed surprisingly high values of typically several tens of mm Hg. Clinical measurements of mitoPO2 are possible as demonstrated by cutaneous measurements in healthy volunteers. Applications of PpIX-TSLT in anesthesiology and intensive care medicine might, e.g., be monitoring mitoPO2 as a resuscitation end point, targeting oxygen homeostasis in the critically ill, and assessing mitochondrial function at the bedside. PpIX-TSLT likely also has applications in other fields also, e.g., providing an oxygen-related feedback signal in photodynamic therapy of malignant tumors.