To clarify the oxygen transport across the microvessels and oxygen distribution in the skeletal muscle, we have newly designed an intravital laser microscopy with oxygen-dependent quenching of phosphorescence technique to determine both the microvascular and the tissue pO2. After injecting the phosphorescent probe into systemic blood, phosphorescence excited by a N2/dye pulse laser was detected with a photomultiplier over a 10 μm in diameter area. In vivo oxygen tension (pO2) measurements revealed the efficiency of this method. This method was then used to measure the pO2 of microvessels with different diameters (40-130 μm) and of interstitial spaces in rat cremaster muscle. These measurements showed a significant reduction of pO2 levels in arterioles of different orders and the presence of large pO2 gradients at the blood/tissue interface of arterioles. These findings suggest that oxygen can be released from larger arterioles and O2 consumption of rat cremaster muscle might be high.