SummaryThe reducing power of glutathione, expressed by its reduction potential E GSH , is an accepted measure for redox conditions in a given cell compartment. In the endoplasmic reticulum (ER), E GSH is less reducing than elsewhere in the cell. However, attempts to determine E GSH (ER) have been inconsistent and based on ineligible assumptions. Using a codon-optimized and evidently glutathione-specific glutaredoxin-coupled redox-sensitive green fluorescent protein (roGFP) variant, we determined E GSH (ER) in HeLa cells as 220864 mV (at pH 7.0). At variance with existing models, this is not oxidizing enough to maintain the known redox state of protein disulfide isomerase family enzymes. Live-cell microscopy confirmed ER hypo-oxidation upon inhibition of ER Ca 2+ import. Conversely, stressing the ER with a glycosylation inhibitor did not lead to more reducing conditions, as reported for yeast. These results, which for the first time establish the oxidative capacity of glutathione in the ER, illustrate a context-dependent interplay between ER stress and E GSH (ER). The reported development of ER-localized E GSH sensors will enable more targeted in vivo redox analyses in ERrelated disorders.