A putative, Na + -dependent Mg 2 + transport pathway controls the intracellular free Mg 2 + concentration ([Mg 2 + ] i ) in various mammalian cells. The characteristics of this Mg 2 + transport pathway have not been clarified. Herein, we examined the regulatory mechanism of Na + -dependent Mg 2 + efflux in renal epithelial NRK-52E cells. Mg 2 + removal from the extracellular bathing solution induced an Na + -dependent [Mg 2 + ] i decrease in Mg 2 + (5 mM)-loaded cells but not in control cells. Amiloride inhibited the [Mg 2 + ] i decrease in a dose-dependent manner (IC 5 0 =3 μM). Similarly, atomic absorption spectrophotometry showed that Mg 2 + removal decreased intracellular Mg 2 + content, while it increased Na + content. Calphostin C (1 μM), a protein kinase C inhibitor, and genistein, a tyrosine kinase inhibitor (10 μM), blocked the [Mg 2 + ] i decrease. The [Mg 2 + ] i decrease was accompanied by an increase in intracellular nitric oxide (NO) and cyclic GMP contents. (E)-4-methyl-2-[(E)-hydoxyimino]-5-nitro-6-methoxy-3-hexenamide (0.1 mM), an NO donor, and 8-bromo-cyclic GMP (0.1 mM), a membrane-permeable cyclic GMP analogue, accelerated the [Mg 2 + ] i decrease. In contrast, N G -monomethyl-l-arginine (l-NMMA, 0.1 mM), an NO competitive inhibitor, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 μM), an NO-sensitive guanylate cyclase inhibitor, significantly blocked the [Mg 2 + ] i decrease. These results indicate that a decrease in extracellular Mg 2 + concentration induces the production of NO and cyclic GMP, which leads to the up-regulation of Na + -dependent Mg 2 + efflux.