In the present study, we show that a combination of sulindac and arsenic trioxide (ATO) induces more extensive apoptosis than either drug alone in H1299 human non-small cell lung carcinoma (NSCLC) cells. Treatment with sulindac/ATO triggered three major apoptotic signaling events, namely, collapse of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. Furthermore, the sulindac/ATO combination induced reactive oxygen species (ROS) generation, and the antioxidant, N-acetyl-L-cysteine, blocked this apoptotic signaling. The c-Jun NH(2)-terminal kinase (JNK) was activated downstream of ROS production in H1299 cells. Blockage of JNK by pretreatment with SP600125, a pharmacological inhibitor, or transfection with dominant-negative (DN) JNK1 vectors abrogated sulindac/ATO-induced apoptosis, as evident from the disruption of caspase activation. Interestingly, a slower migrating Bcl-xL band was observed on immunoblots after treatment of cells with sulindac/ATO. The band was absent upon the treatment of cell lysates with lambda protein phosphatase. Moreover, confocal microscopy findings disclose that active JNK translocates to mitochondria. Treatment with SP600125 and transfection with DN-JNK blocked Bcl-xL phosphorylation, suggesting that JNK plays an important role in sulindac/ATO-induced Bcl-xL phosphorylation. In conclusion, in H1299 human NSCLC cells, sulindac and ATO synergistically induce a high degree of apoptosis, which is mediated by the ROS-dependent JNK activation pathway via Bcl-xL phosphorylation.