We injected acetylcholine (ACh), the neurotransmitter of the parasympathetic nervous system, into the coronary arteries of 28 patients with variant angina. Injection of 10 to 80 gg ACh into the coronary artery responsible for the attack induced spasm together with chest pain and ST segment elevation or depression on the electrocardiogram in 30 of the 32 arteries of 25 of the 27 patients. The injection of 20 to 100 gg ACh into the coronary artery not responsible for the attack in 18 patients resulted in various degrees of constriction in most of them, but no spasm in any of them. After intravenous injection of 1.0 to 1.5 mg atropine sulfate, the injection of ACh into the coronary artery responsible for the attack did not induce spasm or attack in any of the nine coronary arteries injected in eight patients. We conclude that the intracoronary injection of ACh induces coronary spasm and attack in patients with variant angina and that the activity of the parasympathetic nervous system may play a role in the pathogenesis of coronary spasm. We also conclude that the intracoronary injection of ACh is a useful test for provocation of coronary spasm.