Introduction The ionic mechanism underlying heart rate (HR) acceleration by catecholamines is incompletely understood. It is widely accepted that cAMP-dependent regulation of HCN4 channels and the activation of the Na+/Ca2+ exchanger by ryanodine receptors mediated Ca2+ release are the predominant mechanisms of HR acceleration. However, experimental evidences demonstrate that none of these mechanisms is essential for sympathetic regulation of HR, suggesting that other ones could be involved in the positive chronotropic effect of β-adrenoceptors. The sinoatrial node expresses two distinct L-type Ca2+ channel isoforms, Cav1.2, which controls myocardial contractility and Cav1.3. Objective. We previously demonstrated that Cav1.3 channels play a major role in the generation of sinoatrial pacemaker activity and impulse conduction. Here we study the role of Cav1.3 in the acceleration of HR by catecholamines. Method We crossed mice in which Cav1.2 channels are rendered insensitive to dihydropyridines (DHP) (Cav1.2DHP-/-), with mice lacking cAMP dependent regulation of HCN4 (HCN4-CNBD). Cav1.2DHP-/-/ HCN4-CNBD mice enable selective inhibition of Cav1.3 by DHP amlodipine (3 µM) or nifedipine (3 µM), or activation by BayK8644 (0.5 µM), in a genetic background lacking cAMP dependent activation of HCN4. We studied HR regulation ex-vivo using the isolated Langendorff -perfused heart. We also used CaV1.3-flex KI/Hcn4-CreERT2 KI mice, in which CaV1.3 is specifically deleted in HCN4 positive cells. Hearts were perfused under ivabradine (3 µM) to block HCN4 current in presence of the β-adrenergic agonist adrenaline (ADR, 0.3 µM). Results In n=8 Cav1.2DHP-/-/HCN4-CNBD hearts, adrenaline failed to significantly increase HR after selective Cav1.3 inhibition by amlodipine (AMLO:207±19bpm vs AMLO+ADR:220±17bpm n=8 p>0.05 respectively), showing that inhibition of Cav1.3 in the absence of cAMP-dependent regulation of HCN4 channels prevented the positive chronotropic response of the heart and suggesting that catecholaminergic regulation of HR was reliant on Cav1.3 in this genotype. Moreover, the CaV1.3-flex KI/Hcn4-CreERT2 KI mice confirmed these results (IVA:113±25bpm vs IVA+ADR:118±26bpm, n=8 p>0.05 respectively). Consistently, activation of CaV1.3 by BayK8644 in Cav1.2DHP-/-/HCN4-CNBD hearts increased the HR similarly to ADR in the same hearts (ADR: +33.1±7.4% vs BayK: +28.6±4.9% n=8 p>0.05), suggesting that in the absence of cAMP-dependent regulation of HCN4, β-adrenergic regulation of CaV1.3 channels is sufficient to explain the increase in HR by catecholamines. Conclusion Our study thus identifies, for the first time, Cav1.3 channels as key effectors of β-adrenergic regulation of heart rate acceleration.