The perforated-patch, whole-cell voltage-clamp technique was used to characterize a hyperpolarization-activated inward current (if) in cultured pacemaker cells isolated from the rabbit sinoatrial (S-A) node. A modified Tyrode solution was used to block interfering currents. After correcting for uncompensated series resistance, leakage current and liquid-junction potentials between the pipette and bath, we obtained the following values for the maximum conductance and reversal potential of (i(f)): 16.2 +/- 2.8 nS or 0.26 +/- 0.07 nS/pF (0.37 +/- 0.10 mS/cm2); and -27.7 +/- 1.2 mV (n = 10). In the modified Tyrode solution, the threshold for activation of i(f) and the half-activation potential and slope factor for Boltzmann fits were -66 +/- 1 mV (n = 53), -77.2 +/- 2.0 mV and 4.2 +/- 0.4 mV (n = 16), respectively. The time course of i(f) was best fit by a sum of two exponentials. Between -110 and -5 mV, time constants ranged from 0.06 to 0.42 s for the rapid component and from 0.65 to 4.39 s for the slow component. Using these characteristics of i(f) and a short segment of spontaneous firing as the membrane potential, we computed the expected time course of i(f) during normal pacemaker activity. It reached a maximum of -3 pA during the diastolic depolarization and +4 pA during the action potential. We conclude that most of the characteristics of i(f) in cultured S-A node cells are similar to those measured previously in freshly isolated pacemaker cells.