Mg2+ buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na+/Mg2+ transporter by using a newly developed Mg2+ indicator, KMG-20, and also a Na+ indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg2+ concentration ([Mg2+](i)). The rate of decrease of [Mg2+](i) was slower in a Na+-free extracellular medium, suggesting the coupling of Na+ influx and Mg2+ efflux. Na+ influxes were different for normal and imipramine-(a putative inhibitor of the Na+/Mg2+ transporter) containing solutions. FCCP induced a rapid increase in [Na+](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg2+](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg2+ depends on the Na+/Mg2+ transporter in PC12 cells.