The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co(2+)-substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.