We have developed a new phosphorescent probe, PdTCPPNa 4 , whose luminescence properties are affected by local variations of intracellular oxygen tension (PO 2). Spectrofluorometric measurements on living human umbilical venous endothelial cells loaded with this molecule show that a decrease in extracellular oxygen tension induces a decrease of PO 2 , illustrating the phenomenon of oxygen diffusion and validating the use of this probe in living cells. Moreover, KCN-or 2,4-dinitrophenol-induced modifications of respiration do not lead to detectable PO 2 variations, probably because O 2 diffusion is sufficient to allow oxygen supply. On the contrary, activation by acetylcholine or endothelial nitric oxide synthase (eNOS), which produces NO while consuming oxygen, induces a significant decrease in PO 2 , whose amplitude is dependent on the acetylcho-line dose, i.e., the eNOS activity level. Hence, activated cytosolic enzymes could consume high levels of oxygen which cannot be supplied by diffusion, leading to PO 2 decrease. Other cell physiology mechanisms leading to PO 2 variations can now be studied in living cells with this probe.