Despite its very potent vasodilating action in vivo, acetylcholine (ACh) does not always produce relaxation of isolated preparations of blood vessels in vitro. For example, in the helical strip of the rabbit descending thoracic aorta, the only reported response to ACh has been graded contractions, occurring at concentrations above 0.1 µM and mediated by muscarinic receptors^1,2. Recently, we observed that in a ring preparation from the rabbit thoracic aorta, ACh produced marked relaxation at concentrations lower than those required to produce contraction^3,4 (confirming an earlier report by Jelliffe⁵). In investigating this apparent discrepancy, we discovered that the loss of retaxation by ACh in the case of the strip was the result of unintentional rubbing of its intimai surface against foreign surfaces during its preparation. If care was taken to avoid rubbing of the intimai surface during preparation, the tissue, whether ring, transverse strip or helical strip, always exhibited relaxation to ACh, and the possibility was considered that rubbing of the intimai surface had removed endothelial cells⁴. We demonstrate here that relaxation of isolated preparations of rabbit thoracic aorta and other blood vessels by ACh requires the presence of endothelial cells, and that ACh, acting on muscarinic receptors of these cells, stimulates release of a substance(s) that causes relaxation of the vascular smooth muscle. We propose that this may be one of the principal mechanisms for ACh-induced vasodilation in vitio. Preliminary reports on some aspects of the work have been reported elsewhere^4,6.