We have developed a new phosphorescent probe, PdTCPPNa4, whose luminescence properties are affected by local variations of intracellular oxygen tension (PO2). 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 PO2, 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 PO2, 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 PO2, whose amplitude is dependent on the acetylcholine 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 decrease. Other cell physiology mechanisms leading to PO2 variations can now be studied in living cells with this probe. (C) 2002 Elsevier Science.