Pacemaker activity of the sino-atrial node (SAN) constitutes a fundamental physiological function. We demonstrate that SAN pacemaker activity is regulated by an unexpected mechanism involving stimulation by L-type Ca_v1.3 channels of Ca²⁺ release from ryanodine receptors (RYRs). In individual SAN pacemaker myocytes, genetic loss or specific pharmacological inhibition of Ca_v1.3 channels strongly reduced diastolic local Ca²⁺ release events (LCRs) and blunted the positive chronotropic response of spontaneous intracellular Ca²⁺ transients to maximal b-adrenergic receptor activation. Consistent with the view that Ca_v1.3 channels modulate the activity of RyRs, pacemaker activity and LCRs of Ca_v1.3^-/- SAN myocytes showed reduced sensitivity to ryanodine. In Ca_v1.3^-/- SAN myocytes, normal pacemaking could be observed only by perfusion of a low concentration of caffeine that conditioned RYR-dependent Ca²⁺ release. In vivo heart rates of Ca_v1.3^-/- mice showed increased sensitivity to caffeine. Also, while specific pharmacological stimulation of Ca_v1.3 channels increased the number of diastolic LCRs and pacemaking, direct activation of the b-adrenergic receptor pathway could not compensate for the loss of Ca_v1.3 channels. In conclusion, our results show that Ca_v1.3-dependent RyR-mediated Ca²⁺ release plays an important role in pacemaker activity and unravel a previously unexpected physiological mechanism in heart rate determination.