Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammary epithelial cells. To this end, we measured Mg2+ and Ca2+ fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg2+, concomitantly with a rise in intracellular calcium. The increase in intracellular Mg2+ derives from an influx from the extracellular compartment, and does not depend on Ca2+. On the contrary, the increase in intracellular Ca2+ derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg2+ and Ca2+ signals. These findings demonstrate that not only does Mg2+ influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg2+ influx is essential for the Ca2+ signal to occur. (C) 2014 Elsevier Inc. All rights reserved.