A system is described for the in vivo noninvasive measurement of intravascular PO2 at the microscopic level. Under special circumstances the method can also be used to measure interstitial PO2. The PO2 determination is based on the O2-dependent quenching of phosphorescence of palladium-porphyrins bound to albumin. This compound was injected intravenously in the dosage of 30 mg/kg body wt and dissolved in saline to a concentration of 15 mg/ml. The phosphorescence emission was excited by epi-illumination with a strobe xenon arc and measured by a photomultiplier in a well-defined tissue area as small as 15 x 30 microns. A selected portion of the phosphorescence decay was fitted by a single exponential, and the Stern-Volmer equation was used to calculate PO2. Calibration was performed in vitro using saline and blood and was in agreement with previous reports. In vivo observations were made in normal tissue regions from the unanesthetized hamster transparent skin fold chamber preparation. The method allows PO2 determinations, in the range of 0-80 mmHg, in microvessels with diameters of 15-100 microns. Simultaneous transillumination of the tissue also allows measurement of vessel diameter and red blood cell velocity in the same vessels.