An intracellular pool of N-type voltage-operated calcium channels has recently been described in different neuronal cell lines. We have now further characterized the intracellular pool of N-type calcium channels in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cells. Intracellular N-type calcium channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched. 125I-omega-Conotoxin GVIA binding assays on fixed and permeabilized cells revealed that intracellular N-type calcium channels translocate to the plasma membrane in cells exposed to secretagogues (KCl, ionomycin, and phorbol esters). The kinetics, Ca2+ and protein kinase C dependence, and brefeldin A insensitivity of N-type calcium channels translocation were similar to the regulated release of chromogranin B, while no correlation was found with the constitutive secretion of a heparan sulfate proteoglycan. A PC12 subclone deficient in the regulated but not in the constitutive pathway of secretion had a small intracellular pool of N-type calcium channels, and no secretagogue-induced translocation occurred in these cells. Calcium channel translocation was accompanied by a stronger response of Fura-2-loaded cells to depolarizing stimuli, suggesting that the newly inserted channels are functional.