This study introduces redox-sensitive green fluorescent protein (roGFP) indicators for monitoring mitochondrial redox potential in living cells. roGFPs are engineered by substituting cysteine residues in GFP to form disulfide bonds, enabling them to respond to changes in redox potential. roGFP1, targeted to mitochondria in HeLa cells, shows dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH ~8.0 at 37°C). The cytosol of HeLa cells is also unusually reducing but less so than the mitochondrial matrix. roGFP2, with two fluorescence excitation maxima at ~400 and 490 nm, exhibits rapid and reversible ratiometric changes in fluorescence in response to redox potential changes. Crystal structure analyses of roGFP2 reveal a highly strained disulfide bond and localized structural changes in the oxidized state. The redox midpoint potentials of roGFPs were determined using equilibrium constants derived from titration experiments. The results show that roGFP1 has a midpoint potential of -0.288 V and roGFP2 of -0.272 V. The pH dependence of the midpoint potential was investigated, and the redox potential was calculated using the Nernst equation. roGFPs were used to monitor redox changes in single living cells, showing that the mitochondrial matrix is highly reducing. The study highlights the importance of redox homeostasis in cellular processes and the potential of roGFPs as noninvasive tools for monitoring redox status in living cells.This study introduces redox-sensitive green fluorescent protein (roGFP) indicators for monitoring mitochondrial redox potential in living cells. roGFPs are engineered by substituting cysteine residues in GFP to form disulfide bonds, enabling them to respond to changes in redox potential. roGFP1, targeted to mitochondria in HeLa cells, shows dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH ~8.0 at 37°C). The cytosol of HeLa cells is also unusually reducing but less so than the mitochondrial matrix. roGFP2, with two fluorescence excitation maxima at ~400 and 490 nm, exhibits rapid and reversible ratiometric changes in fluorescence in response to redox potential changes. Crystal structure analyses of roGFP2 reveal a highly strained disulfide bond and localized structural changes in the oxidized state. The redox midpoint potentials of roGFPs were determined using equilibrium constants derived from titration experiments. The results show that roGFP1 has a midpoint potential of -0.288 V and roGFP2 of -0.272 V. The pH dependence of the midpoint potential was investigated, and the redox potential was calculated using the Nernst equation. roGFPs were used to monitor redox changes in single living cells, showing that the mitochondrial matrix is highly reducing. The study highlights the importance of redox homeostasis in cellular processes and the potential of roGFPs as noninvasive tools for monitoring redox status in living cells.