Human urinary DNA adducts may be useful surrogate biomarkers to estimate carcinogen exposure and activation, particularly if such adducts are of high selectivity from a specific carcinogen source. In this report, we provided evidence supporting tobacco use and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being the dominant source for 3-methyladenine (3-mA), while nontobacco factors contribute significantly to 7-methylguanine and 1-methyladenine in the urine. Upon confirmation in human urine samples from larger populations in the future, urinary 3-mA may be used to estimate NNK bioactivation in smokers and to facilitate the development of a chemopreventive agent against NNK-induced carcinogenesis.