The I_f and the Cav1.3-mediated L-type Ca²⁺ currents play important roles in the genesis of cardiac automaticity and heart rate (HR). The differential role of f- and Cav1.3- channels in pacemaking is not completely elucidated. More importantly, it is not known if automaticity can persist after inhibition and/or inactivation of both f- and Cav1.3 channels. The effects of I_f inhibition by ivabradine (IVA), the first specific and selective I_f sinus node inhibitor, were investigated on ECG parameters using telemetry in wild-type (WT; n=7) and Cav1.3 knockout (Cav1.3-/-; n=7) mice, spontaneously bradycardic. IVA was administrated once intra-peritoneally (ip bolus) from 0.1 up to 6 mg/kg. In WT and Cav1.3-/- mice, IVA dose-dependently reduced the mean HR (Fig.) without affecting the QRS complex. At 3 mg/kg, mean HR reduction was –23 and –39% in WT and Cav1.3-/- (p<0.01), respectively. Saturation of IVA effect on HR was observed between 3 and 6 mg/kg, suggesting complete and specific in vivo inhibition of I_f at these doses. In Cav1.3-/- mice, intrinsic HR measurement after injection of atropine (0.5 mg/kg ip) and propranolol (1 mg/kg ip) (A+P) to block autonomic input, showed that I_f inhibition by IVA at 3 mg/kg ip did not stop pacemaking but only lowered intrinsic HR from 226±34 bpm at 3 mg/kg to 184±24 bpm with A+P at the same dose (p<0.05). In conclusion, f- and Cav1.3 channels control the mean HR. I_f inhibition lowered both the mean and maximal HR. Contrary to f-channels, Cav1.3 channels do not reduce the maximal HR, but stabilize HR. Most importantly, both normal and intrinsic pacemaker activity can persist in the absence of Cav1.3 channels even after partial inhibition of HCN channels. These observations indicate that multiple mechanisms contribute to cardiac automaticity and, thus to the safety of HR reducing agents such as ivabradine.