Aflatoxin B1 (AFB1) is a mycotoxin produced by some strains of Aspergillus and is a recognized pulmonary and hepatic carcinogen. The most widely accepted mechanism of AFB1 carcinogenicity involves bioactivation to AFB1-8,9-exo-epoxide and binding to DNA to form AFB1-N7-guanine. Another potential cause of DNA damage is AFB1-mediated stimulation of reactive oxygen species formation, leading to oxidation of DNA bases. The objective of this study was to determine the ability of AFB1 to cause oxidative DNA damage in lung cell types of the A/J mouse. The formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells was assessed by high-performance liquid chromatography with electrochemical detection. An approximately 3-fold increase in 8-OHdG formation occurred in both alveolar macrophage and Clara cell preparations isolated from A/J mice 2 h following treatment with a single tumorigenic dose of 50 mg/kg AFB1 ip (n = 3, p < 0.05). Prior treatment with 300 kU/kg polyethylene glycol–conjugated catalase prevented the AFB1-induced increase in 8-OHdG levels in all mouse lung cell preparations (n = 3, p < 0.05). These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB1 carcinogenicity.