Acetylcholine, substance P and nitroglycerin applied intra- and extraluminally to the perfused dog femoral artery segment with endothelium caused depressor responses. Endothelium denudation abolished the responses to acetylcholine and substance P. EC50 ratios of extra- versus intraluminal acetylcholine and substance P were 43 and 79, respectively, whereas those of nitroglycerin did not differ. Physostigmine potentiated the response to extraluminal acetylcholine. Acetylcholine seems to be degraded partly by cholinesterase in the arterial wall. Acetylcholine and substance P applied extraluminally are expected to reach the endothelium and release endothelium-derived relaxing factor.