Reactive oxygen species (ROS) produced by tumor necrosis factor-a (TNF-a) have an important function in cell death by activating c-Jun N-terminal kinase. However, the exact mechanism of mitochondrial ROS production, after TNF-a stimulation, is not clearly understood. In this study, we determined that ROS modulator 1 (Romo1) and B-cell lymphoma-extra large (Bcl-X L ) are directly associated with TNF-a-induced ROS production. In response to TNF-a, TNF complex II, which consists of receptorinteracting protein 1, TNF receptor-associated protein with death domain, TNF receptor-associated factor 2, Fas-associated death domain protein, and pro-caspase-8, binds to the C-terminus of Romo1 located in the mitochondria. Concurrently, Romo1 recruits Bcl-X L to reduce the mitochondrial membrane potential, resulting in ROS production and apoptotic cell death. On the basis of these results, we suggest that Romo1 is a molecular bridge between TNF-a signaling and the mitochondria for ROS production that triggers TNF-a-mediated apoptosis, as well as a novel target in the development of anti-inflammatory agents that block the origin of ROS production. Tumor necrosis factor-a (TNF-a) is a major mediator of inflammation. There are two main pathways in TNF-a signaling. One is the pro-survival/pro-inflammatory pathway that activates NF-kB and MAPK through TNF-a-induced signaling complex I. 1 After binding of the trimeric TNF-a to the cell surface receptor TNF-R1, the silencer of death domain dissociates from TNF-R1 complexes, and the TNF receptor-associated protein with death domain (TRADD) -an adaptor protein -binds to the cytoplasmic domain of the receptor. 2 TRADD recruits additional proteins including TNF receptor-associated factor 2 (TRAF2), receptor-interacting protein 1 (RIP1), and cellular inhibitor of apoptosis proteins to form TNF-a-induced signaling complex I. 3,4 The other TNF-a signaling pathway is the proapoptotic pathway through TNF-ainduced signaling complex II, in which reactive oxygen species (ROS), a caspase cascade, and the mitochondria function as downstream mediators. If TRADD, TRAF2, and RIP1 dissociate from the receptor, they recruit Fas-associated death domain protein (FADD) and pro-caspase-8 to form TNF-a-induced signaling complex II, which is implicated in signaling apoptosis. 5,6 This TNF-a-induced cell death pathway is normally blocked by concomitant activation of NF-kB.ROS generation, after TNF-a binding, has been reported to be involved in both cell survival and cell death, and the main source of ROS generation that contributes to TNF-a-induced cell death is the mitochondrion. 7-9 However, another study reported that NADPH oxidase is the source of ROS generation after TNF-a treatment. 10 ROS are known to contribute to cell death by inducing mitochondrial membrane permeabilization and sustaining c-Jun N-terminal kinase (JNK) activation. 11,12 JNK is activated by the activation of apoptosis signal-regulating kinase-1 or by ROS-mediated inactivation of MKPs. 13,14 There are several reports regarding the mech...