A new protocol has been developed for selective removal of the endothelium from the arteries of the perfused rat hind-limb preparation. The hind limb was perfused with oxygenated Krebs-Ringer bicarbonate solution with a combination of high flow and timed air bubbles (2 minutes at high flow followed by 5 minutes of air bubbles, followed by a further 2 minutes at high flow). Representative vessels at different diameters of the arterial tree–-the femoral artery, the artery supplying the extensor hallucis proprius muscle, and the arteries, arterioles, and capillaries withinthe hallucis proprius muscle–-were taken for examination by transmission and scanning electron microscopy. A graded degree of damage occurred along the length of the hind-limb vasculature, with endothelial cells having been completely removed from the femoral artery, removed from or severely damaged in the artery supplying the hallucis proprius muscle, and partially damaged in arteries within the hallucis proprius muscle. However, no damage occurred to the endothelial cells of the arterioles and capillaries within the hallucis proprius muscle. The integrity of the vascular bed after endothelial removal according to the above protocol was confirmed by the demonstration of no diminution, but in fact an increase, in contractile responses to bolus injections of α,β-methylene ATP, resulting in a shift to the left of the dose-response curve. The viability was further confirmed by the fact that the conductance (flow/perfusion pressure) of the preparation during the periods of high flow was no different before and after removal of the endothelium. In conclusion, the high flow/air bubbles/high flow protocol can be used to selectively remove endothelial cells from the arteries of the rat innd-limb preparation without damage to the endothelium of arterioles and capillaries or to smooth muscle cells.