The study of ischemia and reperfusion injury in the extremity has been hampered by lack of an accurate method of measuring skeletal muscle injury. We used a bilateral isolated in vivo canine gracilis muscle model in 15 anesthetized dogs. The experimental muscles had 4, 6, or 8 hours of ischemia and 1 hour of reperfusion. The contralateral gracilis muscle served as a control. Technetium 99m pyrophosphate (99mTc-PYP), an agent which localizes in injured muscle cells, was used to quantitate canine skeletal muscle damage. After 6 hours of ischemia and 1 hour of reperfusion, there was a significant increase of 215% of 99mTc-PYP uptake in the experimental vs the control muscle. Experimental muscle uptake was 8% greater than control after 4 hours and 405% more after 8 hours of ischemia and reperfusion. Segmental distribution of 99mTc-PYP uptake showed localization to be greatest in the middle of the muscle at the entry site of the gracilis artery. Electron microscopic evaluation also documented this area to have undergone the most severe injury. Distal portions of the muscle did not show increased damage. Our results show that 99mTc-PYP effectively quantitates skeletal muscle ischemia and reperfusion injury. The pattern of 99mTc-PYP uptake suggests that considerable injury is caused during reperfusion.