We developed an isolated rabbit atrial preparation which responds consistently and reproducibly to brief, submaximal stimulation of the autonomic nerves contained in it. In 6 of 11 preparations in the presence of propranolol (1 mg/liter), the time course of changes in the atrial rate following 120 msec vagal stimulation was bimodal. The maximal slowing occurred at 0.64 +/- 0.16 second, and the peak secondary slowing occurred at 2.3 +/- 1.0 seconds. An acceleratory component occurred between the first and second peaks between 0.8 and 1.6 seconds. The total time course of vagal effect lasted for 5.0 +/- 2.0 seconds. These changes in rate could not be explained by shifts in the location of the primary pacemaker. The acceleratory component was due to a 4.7 +/- 2.0 (SD) mV depolarization of the maximum diastolic membrane potential of the primary pacemaker of the sinus node which lasted for 1.8 +/- 0.3 seconds. Following vagal stimulation, there was an increase of 0.2 mM in the activity of potassium in the extracellular space recorded with a potassium-sensitive electrode; this peaked between 1.4 and 2.5 seconds and cleared with an exponential time course. The halftimes for recovery ranged between 2.8 and 4.6 seconds. The initial peak slowing of the bimodal time course and the acceleratory component therefore appear to be direct effects of acetylcholine. The secondary slowing occurs after acetylcholine presumably has been inactivated and occurs coincidently with the accumulation of potassium in the extracellular space.