Zinc plays a critical role in many fundamental cellular processes, acting as a Lewis acid catalyst in numerous enzymes, having a structural function in DNA binding proteins and acting as a modulator in neurotransmission (1-3). At the same time, low nanomolar concentrations of free Zn2+ can be cytotoxic, rendering zinc homeostasis a delicate balance that is not well understood. We developed a toolbox of genetically-encoded Forster Resonance Energy Transfer (FRET)-based sensors that allow monitoring of fluctuations in intracellular free zinc levels. These sensors, called eCALWY-1, -2, -3, -4, -5 and -6, display a large decrease in energy transfer upon Zn2+ binding and have affinities that span the pico- to nanomolar range (Table 1). They were successfully used to determine the cytosolic free Zn2+ concentrations of HEK293 and INS-1(832/13) cells, which were both found to be 0.4 nM. In addition, the sensors can be targeted to subcellular organelles, as was shown for secretory granules in pancreatic beta-cells (4).