Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca(2+)-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca(2+)](i)) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 microM), increased [Ca(2+)](i) in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca(2+) sources. SN56 cells responded to ryanodine by increasing [Ca(2+)](i) and this effect was blocked by dantrolene, an inhibitor of Ca(2+)-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca(2+)](i) induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca(2+)-ATPase inhibitor, we investigated whether the depletion of intracellular Ca(2+) stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca(2+)](i) induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca(2+)](i) by releasing Ca(2+) from intracellular stores in cholinergic cells.