4-Hydroxy-2-nonenal (HNE), one of the main aldehydic compounds released during lipid peroxidation, has been proposed to react with DNA bases in cells. Several classes of DNA lesions involving addition of either HNE or its 2,3-epoxide (epox-HNE) have been identified. In the present work, HPLC associated with tandem mass spectrometry was used to determine the pattern of HNE-induced DNA lesions. First, adducts were quantified within isolated DNA treated with HNE under peroxidizing conditions. The 1,N2-propano-2′-deoxyguanosine adduct of HNE (HNE-dGuo) was found to be the major lesion under all conditions studied. 1,N6-Ethenoadenine and 1,N2-ethenoguanine together with their (1,2-dihydroxyheptyl)-substituted derivatives, which all arise from the reaction of epox-HNE with DNA, were produced in significantly lower yields, even in the presence of 20 mM H2O2. The pyrimidopurinone malondialdehyde-2′-deoxyguanosine adduct was also found to be produced, although in very low yield. Similar results were obtained in cultured human monocytes incubated with HNE, because the HNE-dGuo adduct represented more than 95% of the overall adducts to DNA. In addition, the former lesion was poorly repaired, in contrast to 1,N2-ethenoguanine and, to a lesser extent, 1,N6-ethenoadenine. Altogether, these results suggest than HNE-dGuo may represent the best biomarker of the genotoxic effects of HNE.