We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H2O2) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H2O2-sensing protein, OxyR. Using HyPer we monitored H2O2production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H2O2occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (ΔΨ) and a change in cell shape. We also observed local bursts in mitochondrial H2O2production during ΔΨ oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H2O2increase upon physiological stimulation. Using HyPer we detected temporal increase in H2O2in the cytoplasm of PC-12 cells stimulated with nerve growth factor. © 2006 Nature Publishing Group.