Formaldehyde is considered carcinogenic to humans by the IARC, but there are no previous reports of formaldehyde-DNA adducts in humans. In this study, we used liquid chromatography-electrospray ionization-tandem mass spectrometry to quantify the formaldehyde-DNA adduct N6-hydroxymethyldeoxyadenosine (N6-HOMe-dAdo) in leukocyte DNA samples from 32 smokers of ≥10 cigarettes per day and 30 nonsmokers. Clear peaks coeluting with the internal standard in two different systems were seen in samples from smokers but rarely in nonsmokers. N6-HOMe-dAdo was detected in 29 of 32 smoker samples (mean F SD, 179 F 205 fmol/μmol dAdo). In contrast, it was detected in only 7 of 30 nonsmoker samples (15.5 ± 33.8 fmol/Mmol dAdo; P < 0.001). The results of this study show remarkable differences between smokers and nonsmokers in levels of a leukocyte formaldehyde-DNA adduct, suggesting a potentially important and previously unrecognized role for formaldehyde as a cause of cancer induced by cigarette smoking.