Lipids are known to be major targets of oxidative stress in cells. In addition to deleterious effects on membranes and various cellular processes, lipid peroxidation has been proposed to be an indirect genotoxic pathway. Indeed, reactive aldehydes produced upon degradation of lipid hydroperoxides may add to DNA bases. In the present work, we investigated the DNA damaging properties of exogenously added 4-hydroxy-2(E)-nonenal (HNE) in human THP1 monocytes. To provide quantitative data on the possible role of HNE in oxidative genotoxicity, we applied an accurate HPLC-MS approach to the quantification of HNE adducts to DNA and of HNE conjugates to glutathione (HNE-GSH), the product of the major detoxification pathway of HNE in cells. We confirmed that GSH was more reactive than DNA toward HNE in cells, with a ratio of 25000 between the amounts of HNE-GSH and DNA adducts. In addition, we found that the conjugate of HNE to cysteine was produced in much lower yield than HNE-GSH, while that of N-acetylcysteine could not be detected. We also observed that a decrease in the GSH content resulted in the favored formation of DNA lesions. If our data based on an intense and short exposure to HNE can be extended to an in vivo situation where low concentrations of HNE are produced on a long time scale, the present results suggest that although the amount of DNA adducts is low upon treatment by exogenous HNE, their formation could be favored upon oxidative stress. Indeed, this last process leads to concomitant consumption of GSH by oxidation and induction of lipid peroxidation.