The calcium-sensitive fluorescent probe, quin 2, was employed to investigate the cholinergic receptor regulation of cytosolic free calcium concentration [( Ca2+]i) in isolated rat submandibular acinar cells. Cholinergic receptor stimulation results in a marked and sustained elevation [Ca2+]i. The major component of this response is shown to be due to activation of a calcium influx pathway. There is no evidence in K+-depolarized cells of any voltage-gated calcium influx. Acetylcholine failed to activate the calcium influx pathway in cells bathed in Na+-free solutions. The cholinergic receptors thus regulate a voltage-insensitive but Na+-dependent transport system for calcium entry in submandibular acinar cells.