Accumulated misfolded proteins in endoplasmic reticulum (ER) activate ER stress signaling pathways. Here we identified the ER factors that generate ROS molecules. After mouse NIH3T3 cells were treated with either tunicamycin or thapsigargin, oxidative stress was induced. We found inducible nitric oxide synthase (iNOS) was involved in the generation of ROS induced by ER stress. When thapsigargin-treated cells were pre-treated with iNOS inhibitors 1400W or L-canavanine, their ER stress-induced oxidative stress was almost totally abolished. This effect was not seen in the cells treated with tunicamycin. Therefore, iNOS appears to mediate the ER stress subpathway caused by Ca2+ efflux. To the contrary, after we treated the cells with the 26S proteasome inhibitors lactacystin or MG-132, the UPR-induced oxidative stress dramatically increased, indicating that clearing misfolded proteins from the ER lumen reduced the oxidative stress. Therefore, the oxidative stress induced by ER stress signaling is mediated through both iNOS-dependent and -independent subpathways. (c) 2007 Elsevier Inc. All rights reserved.