Acrolein, the simplest α,β-unsaturated aldehyde, is present in relatively large quantities in cigarette smoke, and several studies have raised the possibility of it being a major etiological agent for smoking-related lung cancer. Acrolein reacts directly with DNA to form primarily Acr-dGuo adducts, which serve as important biomarkers for the assessment of exposure to acrolein and its potential role in smoking-related lung cancer. In this study, we developed an ultrasensitive and low-artifact method using liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry to quantitate Acr-dGuo adducts in normal lung tissue DNA obtained at surgery from lung cancer patients who never smoked and from those who continued smoking until surgery, as confirmed by urinary total cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. This provides a direct comparison of Acr-dGuo levels in human lung tissue as a result of cigarette smoking versus other etiological causes. There was no significant difference between the total Acr-dGuo levels in smokers (28.5 ± 14.9 adducts/10 9 nucleotides) and nonsmokers (25.0 ± 10.7 adducts/10 9 nucleotides), suggesting rapid removal of acrolein by glutathione conjugation and other detoxification mechanisms. Our results do not support the hypothesis that acrolein is a major etiological agent for cigarette smoking-related DNA damage.