A rapidly activating component of delayed rectifier K+ current (IK,r) was dissected using a selective blocker, E-4031, during the action potential clamp (AP clamp) in rabbit sinoatrial node cells. Application of E-4031 induced a large compensation current, of which amplitude was similar to or larger than the net current during repolarization and was maximum (2.2 +/- 0.2 pA/pF) at -46.0 +/- 1.8 mV (n = 13). During the slow diastolic depolarization, the compensation current gradually decayed and then abruptly decreased at the peak of action potential. The time-dependent change of IK,r was calculated using a mathematical model, in which independent gates of activation and inactivation were assumed based on the whole cell voltage-clamp experiments. The reconstructed IK,r corresponded well with the E-4031-sensitive current measured by the AP clamp method. Partial block of IK,r by E-4031 in spontaneously beating cells decreased the action potential amplitude, maximum rate of rise, and maximum rate of repolarization and induced a positive shift of the maximum diastolic potential. Complete block of IK,r terminated the spontaneous action potential at -37.4 +/- 2.9 mV (n = 3). It is concluded that IK,r plays an essential role in determining the maximum diastolic potential and ensures the firing of the following action potential in sinoatrial node cells.