The ability of the human lung to catalyze genotoxic bioactivation of constituents of diesel exhaust particle (DEP) extract (DEPE) and the identity of the lung enzymes involved in the bioactivation were investigated using human lung tissues obtained from surgical resections. Genotoxicity was determined by lung S9-catalyzed mutagenicity of DEPE constituents to Salmonella typhimurium TA98NR in the Ames test and by DEPE-induced pneumocyte DNA damage response as determined by γH2Ax expression in ex vivo tissues. S9 was prepared from lung explants treated ex vivo with either DEPE to induce pulmonary enzymes (DEPE-S9) or vehicle only (CON-S9). TA98NR served as the tester strain for the purpose of enhancing and minimizing the contribution of lung S9 and Salmonella, respectively, to DEPE bioactivation. DEPE-S9 was 2.2–fold more active than CON-S9 or rat liver S9 in DEPE bioactivation and the bioactivation was inhibited 58, 45, 22, and 16% by α-naphthoflavone, dicumarol, ketoconazole, and ticlopidine, respectively. Alveolar S9 was less active than bronchioalveolar S9 in DEPE bioactivation. DEPE and diesel exhaust particles (DEP) induced γ-pH2Ax expression in pulmonary cells. Pulmonary CYP1A1 and NQO1 were induced by DEPE treatment, with the constitutive and induced CYP1A1 distributed throughout all peripheral lung regions, whereas NQO1 was limited in distribution to bronchiolar epithelium. The results show that the human lung is highly active in catalyzing genotoxic bioactivation of diesel emission constituents and that CYP1A and NQO1 play major roles in the reaction. The findings underscore the usefulness of human lung tissues in studies of the pneumotoxicity potential of chemicals to humans. Environ. Mol. Mutagen. 54:158–171, 2013. © 2013 Wiley Periodicals, Inc.