Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N2-propano-dGuo adducts: (6R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-6- hydroxypyrimido[1,2-a]-purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido- [1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). α-OH-Acr-dGuo is more mutagenic and mainly induces G → T transversions. A recent study demonstrated that acrolein-DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in the human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography- electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from the human lung in the presence of [13C10,15N5]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion and by carrying out reactions with NaOH and NaBH4, which produced N2-(3- hydroxypropyl)-dGuo or 1,N2-(1,3-propano)-dGuo from γ-OH-Acr-dGuo and α-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed, and Acr-dGuo adducts were detected in all samples. Both α-OH- and γ-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16-209 adducts/109 nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in the human lung.