Although normally absent, spontaneous pacemaker activity can develop in human atrium to promote tachyarrhythmias. HL-1 cells are immortalized atrial cardiomyocytes that contract spontaneously in culture, providing a model system of atrial cell automaticity. Using electrophysiologic recordings and selective pharmacologic blockers, we investigated the ionic basis of automaticity in atrial HL-1 cells. Both the sarcoplasmic reticulum Ca++ release channel inhibitor ryanodine and the sarcoplasmic reticulum Ca++ ATPase inhibitor thapsigargin slowed automaticity, supporting a role for intracellular Ca++ release in pacemaker activity. Additional experiments were performed to examine the effects of ionic currents activating in the voltage range of diastolic depolarization. Inhibition of the hyperpolarization-activated pacemaker current, I f, by ivabradine significantly suppressed diastolic depolarization, with modest slowing of automaticity. Block of inward Na+ currents also reduced automaticity, whereas inhibition of T-and L-type Ca++ currents caused milder effects to slow beat rate. The major outward current in HL-1 cells is the rapidly activating delayed rectifier, I Kr. Inhibition of I Kr using dofetilide caused marked prolongation of action potential duration and thus spontaneous cycle length. These results demonstrate a mutual role for both intracellular Ca++ release and sarcolemmal ionic currents in controlling automaticity in atrial HL-1 cells. Given that similar internal and membrane-based mechanisms also play a role in sinoatrial nodal cell pacemaker activity, our findings provide evidence for generalized conservation of pacemaker mechanisms among different types of cardiomyocytes.