We previously demonstrated, using rat PC-12 pheochromocytoma cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF), that neuropeptide Y (NPY) inhibits catecholamine synthesis as well as release. Inquiry into the mechanisms of these inhibitions implicated distinct pathways involving reduction of Ca²⁺ influx through voltage-activated Ca²⁺ channels. In the present investigation the effects of NPY on whole cell Ba²⁺ currents were examined to obtain direct evidence supporting the mechanisms suggested by those studies. NPY was found to inhibit the voltage-activated Ba²⁺ current in NGF-differentiated PC-12 cells in a reversible fashion with an EC₅₀ of 13 nM. This inhibition was pertussis toxin sensitive and resulted from NPY modulation of L- and N-type Ca²⁺ channels. The inhibition of L-type channels was not seen with <1 nM free intracellular Ca²⁺ or when protein kinase C (PKC) was inhibited by chelerythrine or PKC-(1931). Furthermore, the effect of NPY on L-type channels was mimicked by the PKC activator phorbol 12-myristate 13-acetate. These studies demonstrate that, in addition to inhibition of N-type Ca²⁺ channels, in NGF-differentiated PC-12 cells NPY inhibits L-type Ca²⁺ channels via an intracellular Ca²⁺- and PKC-dependent pathway.