Acetaldehyde, an ubiquitous mutagen and carcinogen, could be involved in human cancer etiology. Because DNA adducts are important in carcinogenesis, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to explore the presence in human liver DNA of the major acetaldehyde DNA adduct, N2-ethylidenedeoxyguanosine (1). DNA was isolated and enzymatically hydrolyzed in the presence of NaBH3CN, which quantitatively converts adduct 1 to N2-ethyldeoxyguanosine (N2-ethyl-dGuo, 2). [15N5]N2-Ethyl- dGuo was synthesized and used as an internal standard. Adduct 2 was enriched from the hydrolysate by solid phase extraction and analyzed by LC-ESI-MS/MS. Clear peaks were observed for adduct 2 in analyses of human liver DNA, calf thymus DNA, and rat liver DNA. These peaks were not observed, or were much smaller, when the NaBH3CN step was omitted. When the DNA was subjected to neutral thermal hydrolysis prior to NaBH3CN treatment, adduct 2 was not observed. Control experiments using [13C 2] acetaldehyde demonstrated that adducts 1 and 2 were not formed as artifacts during DNA isolation and analysis. These results strongly indicate that adduct 1 is present in human liver DNA and demonstrate that it can be quantified as adduct 2. Levels of adduct 2 measured in 12 human liver samples were 534 ± 245 fmol/μmol dGuo (mean ± SD). The results of this study establish the presence of an acetaldehyde adduct in human liver DNA and suggest that it is a commonly occurring endogenous DNA adduct.