Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (ICa,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 ICa,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 ICa,L density increases postnatally in Npy+/+ (by 56%), but is unchanged in Npy-/-. Both ICa,L density and action potential duration are significantly greater in adult Npy+/+ than Npy-/- myocytes, whereas ICa,L density is equivalent in neonatal Npy+/+ and Npy-/- myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in ICa,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 ICa,L voltage-dependence. Immunoblot analyses and measurements of maximally activated ICa,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 alpha1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in ICa,L density in cardiac myocytes results from regulation ICa,L properties by NPY.