We have found a specific reaction between ketone 1 and peroxynitrite (ONOO-), rather than other reactive oxygen species and reactive nitrogen species generated in the biological system. On the basis of this reaction, we have successfully developed a new fluorescent probe HKGreen-1, which is highly selective for the detection of peroxynitrite in living cells. Before the oxidation with peroxynitrite, the dichlorofluorescein part is masked and the probe is nonfluorescent. However, upon reaction with peroxynitrite, the fluorophore is released, resulting in strong enhancement in fluorescence intensity.