1. The isolated canine right atrium perfused through the sinus node artery at a constant pressure of 100 mm Hg with blood led from a support dog was suspended in a bath filed with blood and kept at constant temperature. This preparation maintained its constant tension development and rate over 5 hrs in all 5 control experiments. 2. A relatively small amount of acetylcholine (ACh) induced a negative inotropic effect at 0.01 mug and a negative chronotropic effect at 0.1 mug. 3. A relatively large dose of ACh induced a biphasic inotropic response, i.e., initially a negative inotropic response and secondarily a long-lasting positive tropic response. These positive chronotropic and inotropic responses were abolished by propranolol, hexamethonium or tetrodotoxin. 6. In the papillary muscle and atrial muscle preparations isolated from one canine heart, ACh caused negative and positive inotropic effect in both paced papillary and atrial muscle preparations. In contrast to the results obtained with atria, the positive inotropic response of the papillary muscle preparation was completely blocked by treatment with propranolol or tetrodotoxin. 7. From these results, it is suggested that in the canine atrium muscarinic mechanisms predominate over nicotinic ones. This may well be due to the known inhibition of nicotinic responses by stimulation of muscarinic receptors.