Interactions between the endogenous estradiol metabolite 2-ME and histone deacetylase inhibitors (HDACIs) have been investigated in human leukemia cells. Co-administration of sub- or marginally toxic concentrations of 2-ME and SAHA or sodium butyrate in diverse human leukemia cell types resulted in a marked increase in oxidative damage (e.g., generation of reactive oxygen species; ROS), mitochondrial injury (e.g., cytochrome c relase and Bax translocation), caspase activation, and apoptosis. These interactions were also noted in primary human leukemia cells but not in normal bone marrow CD34(+) cells. Synergistic interactions between these agents were associated with inactivation of Akt, and activation of JNK(c-Jun N-terminal kinase). Essentially all of these events were reversed by free radical scavengers such as the MnSOD mimetic TBAP, catalase, and ectopic expression of glutathione peroxidase(GPx). Notably, 2-ME/HDACIs treatment resulted in down-regulation of thioredoxin (Trx), MnSOD, and glutathione peroxidase. Enforced activation of Akt blocked 2-ME/HDACIs-mediated mitochondrial injury, caspase activation, and JNK up-regulation, but not ROS generation. Pharmacologic or genetic (siRNA) interruption of the JNK pathway also significantly attenuated the lethality of this regimen. Together, these findings support a model in which antileukemic synergism between 2-ME and HDACIs stems primarily from induction of oxidative damage, leading in turn to Akt inactivation and JNK activation, culminating in mitochondrial injury and apoptosis. They also raise the possibility that these events may preferentially occur in leukemic versus normal hematopoietic cells.