Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is an endogenous source of α,β-unsaturated aldehydes that react with DNA producing a variety of cyclic adducts. The mutagenic cyclic adducts, specifically those derived from oxidation of ω-6 PUFAs, may contribute to the cancer promoting activities associated with ω-6 PUFAs. ( E)-4-Hydroxy-2-nonenal (HNE) is a unique product of ω-6 PUFAs oxidation. HNE reacts with deoxyguanosine (dG) yielding mutagenic 1, N2-propanodeoxyguanosine adducts (HNE-dG). Earlier studies showed HNE can also be oxidized to its epoxide (EH), and EH can react with deoxyadenosine (dA) forming the well-studied εdA and the substituted etheno adducts. Using a liquid chromatography-based tandem mass spectroscopic (LC-MS/MS) method, we previously reported the detection of EH-derived 7-(1',2'-dihydroxyheptyl)-1, N6-ethenodeoxyadenosine (DHHεdA) as a novel endogenous background adduct in DNA from rodent and human tissues. The formation, repair, and mutagenicity of DHHεdA and its biological consequences in cells have not been investigated. To understand the roles of DHHεdA in carcinogenesis, it is important to develop an immuno-based assay to detect DHHεdA in cells and tissues. In this study we describe the development of monoclonal antibodies specifically against DHHεdA and its application to detect DHHεdA in human cells.