Phototoxicity is a side-effect of in vitro and in vivo oxygen partial pressure (pO(2)) detection by luminescence lifetime measurement methods. Dichlorotris(1,10-phenanthroline)-ruthenium(II) hydrate (Ru(Phen)(3)) is a water soluble pO(2) probe associated with low phototoxicity, which we investigated in vivo in the chick's chorioallantoic membrane (CAM) after intravenous or topical administration and in vitro in normal human coronary artery endothelial cells (HCAEC). In vivo, the level of intravenously injected Ru(Phen)(3) decreases within several minutes, whereas the maximum of its biodistribution is observed during the first 2 h after topical application. Both routes are followed by convergence to almost identical "intra/extra-vascular" levels of Ru(Phen)(3). In vitro, we observed that Ru(Phen)(3) enters cells via endocytosis and is then redistributed. None of the studied conditions induced modification of lysosomal or mitochondrial membranes without illumination. No nuclear accumulation was observed. Without illumination Ru(Phen)(3) induces changes in endoplasmic reticulum (ER)-to-Golgi transport. The phototoxic effect of Ru(Phen)(3) leads to more marked ultrastructural changes than administration of Ru(Phen)(3) only (in the dark). These could lead to disruption of Ca2+ homeostasis accompanied by mitochondrial changes or to changes in secretory pathways. In conclusion, we have demonstrated that the intravenous injection of Ru(Phen)(3) into the CAM model mostly leads to extracellular localization of Ru(Phen)(3), while its topical application induces intracellular localization. We have shown in vivo that Ru(Phen)(3) induces minimal photo-damage after illumination with light doses larger by two orders of magnitude than those used for pO(2) measurements. This low phototoxicity is due to the fact that Ru(Phen)(3) enters endothelial cells via endocytosis and is then redistributed towards peroxisomes and other endosomal and secretory vesicles before it is eliminated via exocytosis. Cellular response to Ru(Phen)(3), survival or death, depends on its intracellular concentration and oxidation-reduction properties.