Nuclear orphan receptor TR3/Nur77/NGFI-B is a novel apoptotic effector protein that initiates apoptosis largely by translocating from the nucleus to the mitochondria, causing the release of cytochrome c. However, it is possible that TR3 translocates to other organelles. The present study was designed to determine the intracellular localization of TR3 following CD437-induced nucleocytoplasmic translocation and the mechanisms involved in TR3-induced apoptosis. In human neuroblastoma SK-N-SH cells and human esophageal squamous carcinoma EC109 and EC9706 cells, 5 microM CD437 induced translocation of TR3 to the endoplasmic reticulum (ER). This distribution was confirmed by immunofluorescence analysis, subcellular fractionation analysis and coimmunoprecipitation analysis. The translocated TR3 interacted with ER-targeting Bcl-2; initiated an early release of Ca(2+) from ER; resulted in ER stress and induced apoptosis through ER-specific caspase-4 activation, together with induction of mitochondrial stress and subsequent activation of caspase-9. Our results identified a novel distribution of TR3 in the ER and defined two parallel mitochondrial- and ER-based pathways that ultimately result in apoptotic cell death.