Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-FF has a lower binding affinity for Ca2+ and is suitable for Ca2+ measurements from 10 to 200 uM. Like the parent Rhod-2 indicator, Rhod-FF is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+.