Muscarinic stimulation induces release of Mg2+ from an intracellular pool in rat sublingual mucous acini (Zhang, G. H., and Melvin, J. E. (1992) J. Biol. Chem. 267, 20721-20727). In the present study we examined the interdependence of Mg2+ mobilization on intracellular Na+ and Ca2+ by monitoring the intracellular free concentrations of Na+ ([Na+]i), Mg2+ ([Mg2+]i), and Ca2+ ([Ca2+]i) using ion-sensitive fluorescent indicators. Gramicidin increased the intracellular concentrations of all three ions. Comparable to agonist-stimulated mobilization of Mg2+, the gramicidin-induced [Mg2+]i increase was independent of extracellular Mg2+ indicating release of Mg2+ from an intracellular pool. Clamping the [Ca2+]i near 30 nM with the Ca2+-selective chelator BAPTA failed to alter the [Na+]i or [Mg2+]i increases generated by gramicidin. In contrast, depletion of intracellular Na+ markedly suppressed the muscarinic-stimulated [Mg2+]i increase, whereas the [Ca2+]i increase was similar to that seen in physiological extracellular Na+. These results revealed that intracellular Mg2+ mobilization did not directly relate to the [Ca2+]i, but required an increase in [Na+]i. Consistent with this hypothesis, increasing [Na+]i by activating Na+ influx via the Na+/H+ exchanger also increased the [Mg2+]i. The Na+/Mg2+ exchange inhibitor quinidine suppressed both the gramicidin- and muscarinic-induced discharge of internal Mg2+. These results suggest that release of Mg2+ from an intracellular pool is mediated by a Na+-dependent Mg2+ transport mechanism in salivary acinar cells.