Cell culture studies demonstrate an increase in cardiac L-type Ca²⁺ current (I_Ca,L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in I_Ca,L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy^−/−) and matched controls (Npy^+/+). Whole-cell voltage clamp demonstrates I_Ca,L density increases postnatally in Npy^+/+ (by 56%), but is unchanged in Npy^−/−. Both I_Ca,L density and action potential duration are significantly greater in adult Npy^+/+ than Npy^−/− myocytes, whereas I_Ca,L density is equivalent in neonatal Npy^+/+ and Npy^−/− myocytes. These data indicate NPY does not influence I_Ca,L prenatally, but the postnatal increase in I_Ca,L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy^+/+ and Npy^−/−, indicating NPY does not influence the developmental change in I_Ca,L voltage-dependence. Immunoblot analyses and measurements of maximally activated I_Ca,L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy^+/+ and Npy^−/− cannot be attributed to altered Ca²⁺ channel α_1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in I_Ca,L density in cardiac myocytes results from regulation I_Ca,L properties by NPY.