Secondhand smoke (SHS) exposure is a known risk factor for lung cancer development in lifelong nonsmokers; however, the mechanistic involvement of SHS in the genesis of this malignancy remains elusive. The present study is the first comprehensive investigation of SHS mutagenicity in vivo, in which we have established the mutagenic effects of SHS in transgenic Big Blue mice, and subsequently found correlations between our experimental findings and those obtained from our analysis of the largest database of mutations in human TP53, which is the most frequently mutated gene in human lung cancer. We demonstrate that whole-body SHS exposure of mice for 5 h/d, 5 d/wk for a duration of 2 or 4 mo elicits a significant mutagenic response in the lung, trachea, and bladder of exposed animals, as reflected by the elevation of background cII mutant frequency in the respective organs. The organ-specific mutagenicity of SHS is most pronounced in the lung and remains persistent both in the lung and bladder of SHS-exposed animals after a 1-mo recovery in clean air. The induced cII mutagenesis in the lung of SHS-exposed mice perfectly recapitulates our analysis of the TP53 mutations in human lung cancer in nonsmokers. Remarkably, the relative frequencies of all types of mutations in the TP53 gene of nonsmokers' lung tumors and in the cII transgene of lung cellular DNA from SHS-exposed mice are indistinguishable from one another. We provide the first verification of a mechanistic mode of action for SHS of relevance for carcinogenesis and the first experimental evidence linking SHS exposure to lung cancer in nonsmokers.