Background: Alcohol consumption is one of the top 10 risks for the worldwide burden of disease and an established cause of head and neck cancer, as well as cancer at other sites. Acetaldehyde, the major metabolite of ethanol, reacts with DNA to produce adducts, which are critical in the carcinogenic process and can serve as biomarkers of exposure and, possibly, of disease risk. Acetaldehyde associated with alcohol consumption is considered "carcinogenic to humans." We have previously developed the technology to quantify acetaldehyde-DNA adducts in human tissues, but there are no studies in the literature defining the formation and removal of acetaldehyde-DNA adducts in people who consumed alcohol. Methods: We investigated levels of N 2-ethylidene-dGuo, the major DNA adduct of acetaldehyde, in DNA from human oral cells at several time points after consumption of increasing alcohol doses. Ten healthy nonsmokers were dosed once a week for three weeks. Mouthwash samples were collected before and at several time points after the dose. N 2-Ethylidene-dGuo was measured as its NaBH 3CN reduction product N 2-ethyl-dGuo by liquid chromatography-electrospray-tandem mass spectrometry. Results: N 2-ethylidene-dGuo levels increased as much as 100-fold from baseline within 4 hours after each dose for all subjects and in a dose-responsive manner (P = 0.001). Conclusion: These results show an effect of alcohol on oral cell DNA adduct formation, strongly supporting the key role of acetaldehyde in head and neck cancer caused by alcohol drinking. Impact: Our results provide some of the first conclusive evidence linking exposure to a lifestyle carcinogen and kinetics of DNA adduct formation in humans.