Acetylcholine caused no relaxation of a rubbed strip from rabbit aorta (RASR) which was precontracted with phenylephrine. When the same strip was taken into epithelium-intact guinea-pig trachea, acetylcholine produced a slow-developing relaxation which was antagonized by atropine but not by propranolol, indomethacin, theophylline or hydroquinone. RASR was not relaxed by acetylcholine when it was taken from epithelium-rubbed trachea. The results indicate that acetylcholine releases vascular smooth muscle relaxant factor from tracheal epithelium.