Formation of intra- and intermolecular disulfide bonds is an essential step in the synthesis of secretory proteins. In eukaryotic cells, this process occurs in the endoplasmic reticulum (ER) and requires an oxidative environment with the action of several chaperones and folding catalysts. During protein folding, Ero1p oxidizes protein disulfide isomerase (PDI), which then directly catalyzes the formation of disulfide bonds in folding proteins. Recent cell-free studies suggest that the terminal electron acceptor in the pathway is molecular oxygen, with the resulting formation of hydrogen peroxide (H(2)O(2)). We report for the first time the measurement of ER H(2)O(2) level in live cells. By targeting a fluorescent protein-based H(2)O(2) sensor to various intracellular compartments, we show that the ER has the highest level of H(2)O(2), and this high concentration is well confined to the lumen of the organelle. Manipulation of the Ero1-Lalpha level--either by overexpression or by siRNA-mediated inhibition--caused parallel changes in luminal H(2)O(2), proving that the activity of Ero1-Lalpha results in H(2)O(2) formation in the ER. We also found that calcium mobilization from intracellular stores induces a decrease in ER H(2)O(2) level, suggesting a complex interplay between redox and calcium signaling in the mammalian ER.