Drinking alcohol is a risk factor for cancers of the oral cavity, pharynx, larynx, and esophagus. Although many studies suggest that acetaldehyde, a major metabolite of orally ingested alcohol, plays a crucial role in cancer initiation, the link between the aldehyde dehydrogenase-2 (ALDH2) genotype and acetaldehyde-derived DNA damage has not yet been explored. We have developed a sensitive and quantitative method for detecting the acetaldehyde-derived DNA adducts, N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG), alpha-S- and alpha-R-methyl-gamma-hydroxy-1,N(2)-propano-2'-deoxyguanosine (alpha-S-Me-gamma-OH-PdG and alpha-R-Me-gamma-OH-PdG), and N(2)-(2,6-dimethyl-1,3-dioxan-4-yl)-deoxyguanosine (N(2)-Dio-dG), by using liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) and stable-isotope internal standards. We determined the DNA adducts in 44 blood DNA samples from Japanese alcoholic patients. The levels of three acetaldehyde-derived DNA adducts, N(2)-Et-dG, alpha-S-Me-gamma-OH-PdG, and alpha-R-Me-gamma-OH-PdG, were significantly higher in alcoholics with the ALDH2 1/2 2 genotype compared to those with the ALDH2 1/2 1 genotype. N(2)-Dio-dG was not detected in any of the DNA samples analyzed. These results provide molecular evidence that the ALDH2 genotype affects the genotoxic damage caused by acetaldehyde.