Intracellular roles are commensurate with its abundance in the cell cytoplasm. However, little is known about subcellular dynamics, primarily due to the lack of suitable -selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a -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 . 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 [; ] and its kinetic properties () approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic increase provoked by application of 50 mM 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 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 .