Metabolic activation of the proximate carcinogen benzo[a]pyrene-7,8-trans-dihydrodiol (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) leads to B[a]P-7,8-dione that is both electrophilic and redox-active. B[a]P-7,8-dione generates reactive oxygen species resulting in oxidative DNA damage in human lung cells. However, information on the formation of stable B[a]P-7,8-dione-DNA adducts in these cells is lacking. We studied stable DNA adduct formation of B[a]P-7,8-dione in human lung adenocarcinoma A549 cells, human bronchoalveolar H358 cells, and immortalized human bronchial epithelial HBEC-KT cells. After treatment with 2 μM B[a]P-7,8-dione, the cellular DNA was extracted from the cell pellets subjected to enzyme hydrolysis and subsequent analysis by LC-MS/MS. Several stable DNA adducts of B[a]P-7,8-dione were only detected in A549 and HBEC-KT cells. In A549 cells, the structures of stable B[a]P-7,8-dione-DNA adducts were identified as hydrated-B[a]P-7,8-dione-N(2)-2'-deoxyguanosine and hydrated-B[a]P-7,8-dione-N1-2'-deoxyguanosine. In HBEC-KT cells, the structures of stable B[a]P-7,8-dione-DNA adducts were identified as hydrated-B[a]P-7,8-dione-2'-deoxyadenosine, hydrated-B[a]P-7,8-dione-N1- or N3-2'-deoxyadenosine, and B[a]P-7,8-dione-N1- or N3-2'-deoxyadenosine. In each case, adduct structures were characterized by MS(n) spectra. Adduct structures were also compared to those synthesized from reactions of B[a]P-7,8-dione with either deoxyribonucleosides or salmon testis DNA in vitro but were found to be different.