The role of ROS production on DNA damage and potentiation of fludarabine (F) lethality by the HDAC inhibitor LAQ-824 was investigated in human leukemia cells. Pre-exposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40nM) followed by F (0.4µM) dramatically potentiated apoptosis (≥75%). LAQ-824 triggered an early reactive oxygen species (ROS) peak (30′- 3 h), which declined by 6 h, following LAQ-824 induced Mn-SOD2. LAQ-824/F lethality was significantly diminished by either ROS scavengers NAC (N-acetylcysteine) or Mn-TBAP (manganeseIII-tetrakis 4-benzoic acid porphyrin) or ectopic Mn-SOD2 expression and conversely, increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in γ-H2AX which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA binding activity, and downregulated DNA repair genes BRCA1, CHEK1, and RAD51 ). Addition of fludarabine further potentiated DNA damage which was incompatible with cell survival, and triggered multiple pro-apoptotic signals including activation of nuclear caspase 2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analog-mediated DNA damage and lethality in leukemia. Moreover, they demonstrate for the first time the link between HDAC inhibitor-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.