Stimulation of muscarinic (M3) receptors depolarizes pancreatic B-cells by increasing Na+ influx. Here, we measured [Na+]i and [Ca2+]i in B-cell clusters to investigate whether depletion of intracellular Ca2+ pools triggers this unusual transduction pathway for muscarinic receptors. Acetylcholine emptied Ca2+ pools less completely than did the SERCA pump inhibitors, thapsigargin, and cyclopiazonic acid. However, the rise in [Na+]i produced by acetylcholine was not mimicked by thapsigargin or cyclopiazonic acid and was not prevented by previous depletion of Ca2+ pools. Depolarization of B-cells by acetylcholine stimulates Ca2+ influx and steadily increases [Ca2+]i. In the presence of glucose and extracellular Ca2+, B-cells treated with thapsigargin or cyclopiazonic acid displayed large [Ca2+]i oscillations. Subsequent application of acetylcholine was followed by a sustained rise in [Ca2+]i as in untreated cells. In conclusion, intracellular Ca2+ pool depletion does not mediate acetylcholine stimulation of Na+ entry and of subsequent events. We propose that the muscarinic receptors are coupled to Na+ channels in B-cells.