Glucocorticoids are known to promote apoptosis of eosinophils, normal and neoplastic lymphoid cells, and blastic cells in some patients with acute myeloid leukemia. We investigated the biochemical signal transduction pathways, in particular, the generation of reactive oxygen species (ROS) and activation of caspases in dexamethasone (DEX)-induced apoptosis of eosinophils, and we compared them with those in DEX-sensitive myeloid and lymphoid leukemia cell lines. The GC-receptor antagonist completely abolished DEX-induced apoptosis of eosinophils and leukemia cells. Among inhibitors related to the ROS system, diphenylene iodonium (DPI), a nicotinamide adenine dinucleotide diphosphate (NADPH) oxidase inhibitor, strongly inhibited both spontaneous and DEX-induced apoptosis of eosinophils at concentrations as low as 0.2 to 2 mumol/L, while promoting apoptosis of leukemia cells in a dose-dependent manner. Apocynin, another NADPH oxidase inhibitor, and antioxidants did not affect the apoptosis of eosinophils or leukemia cells. DEX treatment did not change intracellular production of O2- and H2O2, and it decreased the extracellular release of O2- in both cells. These results suggest little or no involvement of ROS generation in DEX-induced apoptosis of both cells. Although among peptide-based caspase inhibitors, only z-VAD-FMK, a broad caspase inhibitor, partially inhibited the apoptosis of eosinophils and leukemia cells, DEX treatment increased the activities of caspases 2-, 3-, 6-, and 8-like proteases assessed by colorimetry in both cells, suggesting the involvement of a similar caspase activation pathway in DEX-induced apoptosis in both cells. DPI markedly reduced caspase 3-like activity in eosinophils, while augmenting the activity in leukemia cells, indicating that DPI acts upstream of caspase 3 activation opposingly in both cells. Thus, the action of DPI in eosinophils seems peculiar in respect to apoptosis induction, and DPI appears to exert an influence on unknown targets rather than those involved in NADPH oxidase inhibition.