We studied the mechanism of post-overdrive suppression in superfused rabbit sinus node pacemaker cells. Small specimens of sinus node tissue isolated from rabbit hearts were driven at a fast rate (overdrive) for 10-120 seconds using single sucrose gap methods. During the control perfusion (35 °C Tyrode's solution), overdrive caused a progressive decrease in maximum diastolic potential (MDP), overshoot (OS), and maximum rate of depolarization at phase 0 [dV/dt)max]. After cessation of the overdrive, the rate of diastolic depolarization decreased, and the spontaneous activity was suppressed temporarily (post-overdrive suppression). MDP, OS, (dV/dt)m.«, and the spontaneous activity returned within a few seconds to the level observed before overdrive. Atropine (2 x 10−6g/ml) did not influence the effects of overdrive. After ouabain administration (3 x 10−7g/ml) or in low temperature perfusate (25°C), the effects of overdrive were accentuated, and a marked suppression of spontaneous activity with a long pause of over several seconds was seen following the overdrive. These results suggest that the post-overdrive suppression of sinus node is attributable, at least in part, to ionic shifts following overdrive, and may be potentiated by metabolic dysfunction of pacemaker cells. Circ Res 46: 90-99, 1980