Treatment for 14 to 24 hours with low concentrations of arsenic trioxide (As2O3, 1-4 μM) caused apoptosis in U-937 promonocytes and other human myeloid leukemia cell lines (HL-60, NB4). This effect was potentiated by cotreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin, and the Akt inhibitor Akti5. However, the inhibitors did not increase the toxicity of the mitochondria-targeting drug lonidamine, and the DNA-specific drugs camptothecin and cisplatin, when used under similar experimental conditions as As 2O3. The potentiation of As2O 3-provoked apoptosis involved the increased disruption of mitochondrial transmembrane potential, increased caspase-3 activation and cytochrome c release from mitochondria, increased Bax and Bid activation, and attenuation of 27-kDa heat shock protein (HSP27) expression; the potentiation was prevented by Bcl-2 overexpression. The PI3K/AM inhibitors decreased the intracellular glutathione content, and caused intracellular oxidation, as measured by peroxide accumulation. Cotreatment with subcytotoxic concentrations of hydrogen peroxide increased apoptosis induction by As2O 3. On the other hand, the treatments did not significantly affect glutathione S-transferase π expression and activity. These results, which indicate that glutathione is a target of PI3K/Akt in myeloid leukemia cells, may partially explain the selective increase of As2O3 toxicity by PI3K/Akt inhibitors, and may provide a rationale to improve the efficacy of these inhibitors as therapeutic agents. © 2005 by The American Society of Hematology.