Intracellular Mg2+ roles are commensurate with its abundance in the cell cytoplasm. However, little is known about Mg2+ subcellular dynamics, primarily due to the lack of suitable Mg2+ -selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a Mg2+ -sensitive indicator—MagIC (indicator for Magnesium Imaging in Cell) —composed of a functionalized yellow fluorescent protein (FP) variant fused to a red-emitting FP serving as a reference, thus allowing ratiometric imaging of Mg2+ . MagIC expressed in mammalian cells is homogeneously distributed between the cytosol and nucleus but its fusion with appropriate targeting sequences redirects it to mitochondria or the endoplasmic reticulum. MagIC shows little interference by intracellular Ca2+ [Kd(Mg2+)=5.1 mM; Kd(Ca2+)=4.8 mM] and its kinetic properties (koff=84 s−1) approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic Mg2+ increase provoked by application of 50 mM MgCl2 in the medium. This effect is, however, mimicked by 75 mM KCl or 150 mM D-sorbitol addition, indicating that it is a response to the associated hyperosmotic shock and not to Mg2+ itself. Our results confirm the functionality of MagIC as a useful tool for the long-awaited possibility of prolonged and organelle-specific monitoring of cellular Mg2+.