Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg(2+), MagFRET-1. This sensor is based on the high-affinity Mg(2+) binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg(2+)-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg(2+) affinity (K d = 148 µM) with a 50% increase in emission ratio upon Mg(2+) binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg(2+) affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg(2+) concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg(2+) concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca(2+), this affinity is sufficiently attenuated (K d of 10 µM) to make the sensor insensitive to known Ca(2+) stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg(2+) imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg(2+) sensors could prove very useful in understanding intracellular Mg(2+) homeostasis and signaling.