Discovered as a DNA repair protein, Ape1 has been associated with other functions, notably redox regulation of transcription factors (Ref1 activity). Because deletion of the mouse gene produces embryonic lethality and stable Ape1-deficient cell lines have not been reported, there has been uncertainty about a possible vital cellular function of Ape1. We addressed this issue by using RNA interference (RNAi) in several human cell types. Strong downregulation of Ape1 stopped cell proliferation and activated apoptosis, which was correlated with accumulation of abasic DNA damage. These effects were reversed by expression of yeast Apn1 protein, which is structurally unrelated to Ape1 but shares enzymatic activity in repair of abasic sites (AP endonuclease). Because Apn1 would lack Ref1 activity or the protein interactions of Ape1, we conclude that the AP endonuclease activity is essential for cellular viability. Accumulation of abasic DNA damage from intrinsic sources appears sufficient to trigger cell death when Ape1-mediated repair is deficient.