4-Hydroxy-l-(3-pyridyl)-l-butanone (HPB)-releasing adducts are formed by metabolic activation of N′-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and have been proposed as specific biomarkers for exposure to tobacco smoke. However, in several studies hemoglobin adducts releasing HPB were on average less than threefold higher in smokers compared to nonsmokers. Using an improved analytical method we have recently found a sevenfold difference in DNA adduct levels in the lung from smoking and nonsmoking lung cancer patients. In the present study we extended the determination of HPB-releasing DNA adducts by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) to samples of peripheral lung, lower esophagus and cardia from tumor-free sudden death victims (primarily road traffic accidents, suicide and sudden cardiac arrest). The donors were classified as either current smokers or nonsmokers based on cotinine in either blood or urine (cut-off values for active smoking: >15 ng cotinine/ml blood or >100 ng cotinine/ml urine). Contrary to our expectation, DNA adduct levels (fmol HPB/mg DNA) in lung tissue from tumor-free smokers (N = 32, 92 ± 148) were not significantly different from values in nonsmokers (N = 56, 61 ± 66). The values in tumor-free smokers were on average more than fourfold lower compared to smoking lung cancer patients in our previous study. Adduct levels in the mucosa of esophagus (N = 82; 133 ± 160) and cardia (N = 30; 108 ± 102) of sudden death victims did not show any difference according to the current smoking status. HPB-releasing DNA adduct levels in cardia and esophagus were significantly correlated (N = 29; Spearman r = 0.609; p < 0.001). In contrast, adduct levels in lung did not correlate with either esophagus (77 cases) or cardia (28 cases). Further studies are necessary to elucidate the discrepancies in lung DNA adduct levels in smokers with or without lung cancer and to identify obvious additional sources other than tobacco for these adducts.