Low concentrations of As(2)O(3) (</=1 micromol/L) induce long-lasting remission in patients with acute promyelocytic leukemia (APL) without significant myelosuppressive side effects. Several groups, including ours, have shown that 0.5 to 1 micromol/L As(2)O(3) induces apoptosis in APL-derived NB4 cells, whereas other leukemic cells are resistant to As(2)O(3) or undergo apoptosis only in response to greater than 2 micromol/L As(2)O(3). In this report, we show that the ability of As(2)O(3) to induce apoptosis in leukemic cells is dependent on the activity of the enzymes that regulate cellular H(2)O(2) content. Thus, NB4 cells have relatively low levels of glutathione peroxidase (GPx) and catalase and have a constitutively higher H(2)O(2) content than U937 monocytic leukemia cells. Glutathione-S-transferase pi (GSTpi), which is important for cellular efflux of As(2)O(3), is also low in NB4 cells. Moreover, As(2)O(3) further inhibits GPX activity and increases cellular H(2)O(2) content in NB4 but not in U937 cells. Selenite pretreatment of NB4 cells increases the activity of GPX, lowers cellular H(2)O(2) levels, and renders NB4 cells resistant to 1 micromol/L As(2)O(3). In contrast, concentrations of As(2)O(3) that alone are not capable of inducing apoptosis in NB4 cells induce apoptosis in the presence of the GPx inhibitor mercaptosuccinic acid. Similar effects are observed by modulating the activity of catalase with its inhibitor, aminotriazol. More important from a therapeutic point of view, U937 and HL-60 cells, which require high concentrations of As(2)O(3) to undergo apoptosis, become sensitive to low, clinically acceptable concentrations of As(2)O(3) when cotreated with these GPx and catalase inhibitors. The induction of apoptosis by As(2)O(3) involves an early decrease in cellular mitochondrial membrane potential and increase in H(2)O(2) content, followed by cytochrome c release, caspase 3 activation, DNA fragmentation, and the classic morphologic changes of apoptosis.