Magnesium (Mg 2+ ) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg 2+ transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3 , as major mechanisms of Mg 2+ influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg 2+ . Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg 2+ concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg 2+ or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg 2+ transport system.