Burn & Walker (1954) described experiments carried out in the heart-lung preparation of the dog which indicated that acetylcholine (ACh) was liberated in that preparation without stimulation of the vagus nerves. They observed that the addition of substances like eserine or neostigmine to the blood, even in low concentration, caused a large fall in the rate of the beat. These substances inhibit the destruction of acetylcholine, and since in addition the fall in rate which they caused was abolished by a small amount of atropine, the conclusion was drawn that the effects observed were due to continuous liberation of small amounts of ACh. Briscoe & Burn (1954) demonstrated that the isolated heart of the rabbit formed ACh, and it was therefore reasonable to suppose that the ACh liberated in the heart-lung preparation came from the heart and not from the lungs.Burn, Vaughan Williams & Walker (1955a) showed that in the heart-lung preparation ACh, infused at constant rate, prolonged the P-R interval, reduced the systemic output of blood and produced A-V block. They also showed that the application of a few stimuli to the auricular appendage during the infusion of the appropriate amount of ACh caused auricular fibrillation. In a later paper (1955b) they found that the addition of inhibitors of cholinesterase to the heart-lung preparation without ACh infusion had similar effects. The P-R interval was prolonged, the systemic outflow was reduced and, in sufficient concentration, there was A-V block. When stimulation was applied to the auricle, the rate at which A-V block began was much reduced. Finally, after stimulation at higher rates in the presence of eserine or paraoxon, auricular fibrillation occurred for a length of time which depended on the concentration of the inhibitor. All this evidence gave further support to the view that ACh was liberated by the heart.