Treatment with the anti-leukemic drug arsenic trioxide (As2O3, 1-4 μM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFα. As2O3 plus TNFα increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIPL expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIPL down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As2O3 does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFα-provoked stimulation of p65-NF-κB translocation to the nucleus and the increase in NF-κB binding activity. Treatments with TNFα alone or with As2O3 plus TNFα cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As2O3 plus TNFα-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As2O3 to TNFα-induced apoptosis in promonocytic leukemia cells is an Akt/NF-κB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways. © 2007 Elsevier B.V. All rights reserved.