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Much research on the activity and half-life of endothelium-derived substances has entailed the removal of endothelium from arteries by mechanical or enzymatic processes. It has been observed that the technique used for the removal of arterial endothelium may profoundly affect smooth muscle function and release of prostanoids by the vessel wall. The function and patterns of regeneration of arterial endothelium have been extensively described, but there is a relative paucity of information about the venous endothelium, due in part to the difficulty of its removal. We developed a technique for removal of the endothelium of rabbit femoral veins by passing a stream of air through the lumen of the vessel to dry and remove the endothelium. The effectiveness of endothelium removal was verified by the lack of in vitro reactivity to endothelium-dependent relaxing substances, examination of frozen sections of vessels, labeled with fluorescent-tagged acetylated low-density lipoprotein, with fluorescent light microscopy and scanning electron microscopy of vessel segments. Air drying effectively removed the endothelium and abolished mechanical responses to endothelium-dependent vasodilators but did not affect the function of the smooth muscle. We propose the use of air to remove endothelium from veins to be used to study endothelium-derived factors since this method achieves complete removal of endothelium without causing detectable damage (morphological or functional) to the remainder of the vessel wall.