Endogenous formation of the mutagenic DNA adduct 1,N⁶-ethenoadenine (εA) originates from lipid peroxidation. Elevated levels of εA in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for εA lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair εA lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2^−/− mice indicates that mABH2 is the principal dioxygenase for εA repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of εA lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of εA, comprise the cellular defense against εA lesions. [Cancer Res 2008;68(11):4142–9]