Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat under normal conditions. The atrio-ventricular node (AVN) also displays pacemaker activity and can take over the control the heartbeat upon SAN failure. The molecular bases of AVN automaticity are poorly understood. We have isolated spontaneously beating cells from the mouse AVN region and studied ionic channels and pacemaker activity by employing the whole-cell configuration of the patch-clamp technique. Upon enzymatic isolation, 10% of cells displayed spindle-shaped morphology and regular beating. Spontaneously-active AVN cells displayed intrinsic slower pacing rate compared to SAN cells (180± 15 bpm in n=11 AVN cells vs. 280± 20 bpm in n=7 SAN cells, p<0.05). The maximum pacing rate measured upon stimulation by 100 nM isoproterenol was higher in SAN than in AVN cells (350±10 bpm, vs 240± 20 bpm, p<0.01). The hyperpolarization-activated current, (If) was found in all SAN and AVN cells tested. If density and that of the inward rectifier current (IK1) were respectively lower and higher in AVN (If density:17± 4 pA/pF in AVN and 39± 4 pA/pF in SAN, n=10 p<0.05) that in SAN cells. In three AVN cells tested, If inhibition by 3µM zeneca inhibited pacing by 30%, indicating an important role for this current in AVN automaticity. The density of the L-type Ca2+ current (ICa,L) was lower in AVN than in SAN cells (1.7±0.2 n=6, vs 4.8±0.3, n=5, p<0.05). AVN cells lacking L-type Cav1.3 Ca2+ channels (from Cav1.3-/ mice) had strongly reduced pacemaker activity. Indeed, upon 10 cells investigated, only 4 Cav1.3-/- AVN cells displayed pacemaker activity. Both cardiac-TTX resistant and TTX-sensitive Na+ currents were found in AVN cells. Blocking TTX-sensitive currents by 100 nM TTX significantly reduced pacemaker activity (from 260± 16 bpm to 221± 18 bpm n=8, p<0.05), leaving unaffected the action potential (AP) waveform, while blocking both TTX-sensitive and TTX-resistant channels with 20 µM TTX strongly reduced pacemaking (from 210± 22 bpm to 80 ± 22 bpm, n=8, p<0.01) and affected the AP upstroke and duration. Delayed-rectifier (IKr) current was consistently found in automatic AVN cells. In contrast, no KvLQT1 related (IKs) or transient outward (Ito) current were found. We conclude that both If and Cav3.1 channels play an important role in automaticity of AVN cells. Enhanced expression of IK1 in AVN cells, together with reduced expression of If, ICa,L can in part explain the intrinsic faster pacing in the SAN cells compared to AVN.