We directly examined the role of the Ca_v1.3 (α_1D) Ca²⁺ channel in the sinoatrial (SA) node by using Ca_v1.3 Ca²⁺ channel-deficient mice. A previous report has shown that the null mutant (Ca_v1.3^−/−) mice have sinus bradycardia with a prolonged PR interval. In the present study, we show that spontaneous action potentials recorded from the SA nodes show a significant decrease in the beating frequency and rate of diastolic depolarization in Ca_v1.3^−/− mice compared with their heterozygous (Ca_v1.3^+/−) or wild-type (WT, Ca_v1.3^+/+) littermates, suggesting that the deficit is intrinsic to the SA node. Whole-cell L-type Ca²⁺ currents (I_Ca,Ls) recorded in single isolated SA node cells from Ca_v1.3^−/− mice show a significant depolarization shift in the activation threshold. The voltage-dependent activation of Ca_v1.2 (α_1C) versus Ca_v1.3 Ca²⁺ channel subunits was directly compared by using a heterologous expression system without β coexpression. Similar to the I_Ca,L recorded in the SA node of Ca_v1.3^−/− mutant mice, the Ca_v1.2 Ca²⁺ channel shows a depolarization shift in the voltage-dependent activation compared with that in the Ca_v1.3 Ca²⁺ channel. In summary, using gene-targeted deletion of the Ca_v1.3 Ca²⁺ channel, we were able to establish a role for Ca_v1.3 Ca²⁺ channels in the generation of the spontaneous action potential in SA node cells.