Previous studies suggest that acetylcholine (ACh) is a transmitter released from taste cells as well as a transmitter in cholinergic efferent neurons innervating taste buds. However, the physiological effects on taste cells have not been established. I examined effects of ACh on taste-receptor cells by monitoring [Ca2+]i. ACh increased [Ca2+]i in both rat and mudpuppy taste cells. Atropine blocked the ACh response, butd-tubocurarine did not. U73122 , a phospholipase C inhibitor, and thapsigargin, a Ca2+-ATPase inhibitor that depletes intracellular Ca2+stores, blocked the ACh response. These results suggest that ACh binds to M1/M3/M5-like subtypes of muscarinic ACh receptors, causing an increase in inositol 1,4,5-trisphosphate and subsequent release of Ca2+ from the intracellular stores. A long incubation with ACh induced a transient response followed by a sustained phase of [Ca2+]i increase. In Ca2+-free solution, the sustained phases disappeared, suggesting that Ca2+ influx is involved in the sustained phase. Depletion of Ca2+ stores by thapsigargin alone induced Ca2+ influx. These findings suggest that Ca2+ store-operated channels may be present in taste cells and that they may participate in the sustained phase of [Ca2+]i increase. Immunocytochemical experiments indicated that the M1 subtype of muscarinic receptors is present in both rat and mudpuppy taste cells.