The 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 GFP (roGFP) variant, we determined E(GSH)(ER) in HeLa cells as -208±4 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 hypooxidation 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-targeted E(GSH) sensors will enable more targeted in vivo redox analyses in ER-related disorders.