The view that Ca2+ entry through voltage-dependent Ca2+ channels (VDCC) and through nicotinic receptors for acetylcholine (nAChRs) causes equal catecholamine release responses in chromaffin cells, was reinvestigated here using new protocols. We have made two-step experiments consisting in an ACh prepulse followed by a depolarizing pulse (DP). In voltage-clamped bovine chromaffin cells an ACh prepulse caused a slow-rate release but augmented 4.5-fold the much faster exocytotic response triggered by a subsequent depo- larizing pulse (measured with capacitance and amperometry). If the ACh prepulse was given with mecamylamine or in low external Ca2+, the secretion increase disappeared. This suggests a two-step model for the effects of ACh: (1) meager Ca2+ entry through nAChRs mostly serves to keep loaded with vesicles the secretory machine; and (2) in this manner, the cell is prepared to respond with an explosive secretion of catecholamine upon depolarization. and fast high Ca2+ entry through VDCC. (c) 2007 Published by Elsevier Inc.