The involvement of oxygen in reactive hyperemia following brief periods of local flow arrest was investigated by measuring PO2 under conditions of different ischemic durations of 5, 15, 30, 60, and 300 sec. Spinotrapezius muscles of anesthetized male Sprague‐Dawley rats were used; the muscle was covered with a gas barrier film to isolate it from atmospheric oxygen. Well perfused sites were chosen with one or more arterioles and a capillary network. Phosphorescence quenching microscopy was used to measure interstitial PO2 with a topically applied phosphorescent probe. Localized ischemia was produced by rapidly inflating an airbag over the muscle; reperfusion was initiated by deflating the bag. Baseline PO2 varied between 52 and 60 mmHg. During ischemia, PO2 dropped to 45 mmHg for 5 s ischemia down to 3 mmHg for >;30 s ischemia. Maximum PO2 during the reperfusion phase ranged from 52 to 71 mmHg. The time required for PO2 recovery to baseline ranged from 9 (5 s ischemia) to 122 sec (300 s ischemia). These data exhibited a positive correlation between ischemic duration and the time for PO2 recovery, as well as to the maximum PO2 reached during reperfusion. The data will be used to establish a relationship between ischemic duration and tissue oxygen consumption (VO2) during reperfusion.Support: NHLBI grant HL18292