In sublingual mucous acini, membrane depolarization induces a threefold transient increase in cytosolic free Ca(2+) concentration [(Ca(2+))i]. The underlying mechanism was examined by using the Ca(2+) sensitive fluorescent indicator fura-2. Membrane depolarization with high K+ induced a transient [Ca(2+)]i increase in acini, but not in single acinar cells. Atropine, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the[Ca(2)+]i increase, suggesting the involvement of muscarinic receptor activation. Inhibition of the inositol trisphosphate (IP3)-sensitive Ca(2+) release pathway with S-(diethylamino)-octyl-3,4,5-trimethoxybenzoate prevented the depolarization-induced increase in [Ca(2+)]i. Blockade of nicotinic receptors and L-, N-, and P-type voltage-dependent Ca(2+) channels (hexamethonium, nifedipine, diltiazem, (omega-conotoxin GVIA and omega-agatoxin IVA) did not inhibit the increase in [Ca(2+)]i. However, Cd(2)+ (0.2 mM) blocked >85 percent of the [Ca2+]i increase. The depolarization-induced [Ca(2+)]i increase was also extracellular Ca(2+)-dependent. These results suggest that the membrane depolarization-induced Ca(2+) increase in sublingual acini is mediated by activating Cd(2+)-sensitive, voltage-dependent Ca(2+) channels in nerve terminals associated with the dispersed acini and stimulating release of acetylcholine, which then triggers the [Ca(2+)]i increase in acinar cells.