(A) roGFP2 contains two Cys residues capable of forming an intramolecular disulfide bond in response to changes in intracellular EGSH. The redox equilibrium of roGFP2 with the GSH/GSSG couple occurs through endogenous glutaredoxins and proceeds slowly. (B) Fusion of human Grx1 in Grx1-roGFP2 substantially improved specificity and rate of thiol-disulfide exchange between roGFP2 dithiol and 2GSH/GSSG couple. (C) Replacing Grx1 with Mrx1 in Mrx1-roGFP2 ensured continuous equilibration between roGFP2 and 2MSH/MSSM pair.