Circulation time studies with acetylcholine were performed in 22 unan-esthetized normal dogs and 7 anesthetized open-chested animals. The end point was the transitory inhibition of the sinus node or, occasionally, the A-V junctional tissue. In the unanesthetized dog, records were obtained with the modified Hamilton manometer in order to observe simultaneous blood pressure responses. Circulation time from foreleg to sinus node or A-V junction ranged from 4.0-9.5 secs., averaging 6.7 secs. In the anesthetized animal, the vascular bed was fractionated by injn. of the agent into its various components and recording the events electrocardiographically. The avg. circulation time (to the first dropped beat) from the superior vena cava was 6.7 secs., from the right ventricle 6.0 secs., from the main pulmonary artery 5.2, from the left ventricle, 1.8, from the root of the aorta 1.3, and when injn. was made 2-3 cm. above the sinuses of Valsalva the avg. interval was 3.4 secs. Injn. high in the ascending aorta, transverse aorta or in the carotid arteries yielded no cardiac end point. Injn. into the main pulmonary artery followed by immediate liga-tion of the right coronary artery likewise resulted in no end point on the initial trials, indicating that the agent operated by a direct effect upon the sinus node. The figures obtained -by this method are smaller than those obtained with other procedures for reasons presented in the discussion, and indicate that a more objective and accurate measure of the circulation time is secured with acetylcholine. It is felt that owing to its simplicity and greater objectivity, the acetylcholine method may be superior to other available procedures.