1. Intracellular recordings were made from pacemaker cells lying in the sino-atrial node of guinea-pigs. 2. Low-frequency vagal stimulation slowed the rate of generation of pacemaker action potentials; high-frequency stimulation stopped the generation of action potentials. 3. During vagal stimulation the rate of diastolic depolarization was reduced with the action potential otherwise unchanged: when the heart stopped the membrane potential of pacemaker cells settled to a value positive of the maximum diastolic potential. 4. In contrast, added acetylcholine caused membrane hyperpolarization and shortened the duration of action potentials. 5. The effects of both added acetylcholine and vagally released acetylcholine were abolished by hyoscine. 6. It is suggested that neurally released acetylcholine acts to change the balance between inward and outward current flow during diastole by modifying the properties of existing voltage-dependent channels. In contrast added acetylcholine appears to activate a different set of receptors which increase the potassium conductance of pacemaker cells.