Although correlative studies demonstrate a reduction in the expression of apurinic/apyrimidinic endonuclease/redox effector factor (Ape1/Ref-1 or Ape1) in neural tissues after neuronal insult, the role of Ape1 in regulating neurotoxicity remains to be elucidated. To address this issue, we examined the effects of reducing Ape1 expression in primary cultures of hippocampal and sensory neurons on several endpoints of neurotoxicity induced by H2O2. Ape1 is highly expressed in hippocampal and sensory neurons grown in culture as indicated by immunohistochemistry, immunoblotting and activity. Exposing hippocampal or sensory neuronal cultures to 25 or 50 nM small interfering RNA to Ape1 (Ape1siRNA), respectively, for 48 h, causes a reduction in immunoreactive Ape1 by approximately 65 and 54%, and an equivalent loss in endonuclease activity. The reduced expression of Ape1 is maintained for up to 5 days after the siRNA in the medium is removed, whereas exposing cultures to scrambled sequence siRNA (SCsiRNA) has no effect of Ape1 protein levels. The reduction in Ape1 significantly reduces cell viability in cultures 24 h after a 1-h exposure to 25-300 microM H2O2, compared to SCsiRNA treated controls. In cells treated with SCsiRNA, exposure to 300 microM H2O2 reduced cell viability by 40 and 30% in hippocampal and sensory neuronal cultures, respectively, whereas cultures treated with Ape1siRNA lost 93 and 80% of cells after the peroxide. Reduced Ape1 levels also increase caspase-3 activity in the cells, 2-3-fold, 60min after a 1-h exposure to 100 microM H2O2 in the cultures. Exposing neuronal cultures with reduced expression of Ape1 to 65 microM H2O2 (hippocampal) or 300 microM H2O2 (sensory) for 1h results in a 3-fold and 1.5-fold increase in the phosphorylation of histone H2A.X compared to cells exposed to SCsiRNA. Overexpressing wild-type Ape1 in hippocampal and sensory cells using adenoviral expression constructs results in significant increase in cell viability after exposure to various concentrations of H2O2. The C65A repair competent/redox incompetent Ape1 when expressed in the hippocampal and sensory cells conferred only partial protection on the cells. These data support the notion that both of functions of Ape1, redox and repair are necessary for optimal levels of neuronal cell survival.