Tobacco smoke consists of numerous carcinogens whose effect on lung tumor development includes the induction of mutations in key genes as well as the induction of chromosome instability (CIN). Consequently, carcinogen-induced mouse lung adenocarcinomas (LAC) display many more recurrent site- and chromosome-specific changes in DNA copy number compared with noninduced LAC. Here we identified the Adenylosuccinate synthetase 1 (Adss1) gene located on distal chromosome 12q as a focus of bi-allelic or homozygous deletion (HD) in LAC. HDs of Adss1 were detected in 10 out of 84 carcinogen-induced mouse primary LAC and mouse LAC cell lines. In only four of these cases did the deletions affect either Siva1 or Inverted-formin 2 (Inf2), which immediately flank the Adss1 locus, indicating that Adss1 is a selective target of deletion in LAC. Losses of Adss1 not meeting the quantitative threshold of HD were detected in 36 out of 84 (42.9%) of the mouse tumors and cell lines. A similar frequency of ADSS1 deletion was observed in human LAC cell lines, suggesting relevance in human lung cancer. Adss1 losses were also found to be significantly associated with a more extensive CIN phenotype in the primary mouse tumors. These results implicate ADSS1 inactivation as a novel somatic alteration in lung carcinogenesis, and suggest that its selective deletion in LAC may be triggered by CIN.