Occupational exposures to respirable dusts contaminated with the mycotoxin aflatoxin B1 (AFB1) have been associated with an increased incidence of upper airway tumors. To investigate this possible etiology we compared the abilities of tracheal and lung S9 from rabbit, hamster and rat to activate AFB1 to mutagens in Salmonella typhimurium TA 98. The activation of AFB1 was compared to that of benzo[a]pyrene (B[a]P), a known respiratory carcinogen. These species differ in airway morphology with respect to numbers of metabolically-active non-ciliated tracheal epithelial cells. Tracheas from hamster and rabbit and lung from rabbit were active in converting AFB1 to bacterial mutagens. Trachea from hamster was more efficient in activating AFB1 to mutagens than lung, while rabbit lung was over 4 times more active in converting AFB1 to mutagens than that from trachea. In all cases, AFB1 was more mutagenic than B[a]P. The relative capabilities of trachea to activate AFB1 were in agreement with the ability of cultured tracheas from these species to form to AFB1-DNA adducts. These results demonstrate that AFB1 is activated more efficiently than B[a]P in the lung, and that the metabolic capabilities of airway epithelium to activate AFB1 are not predictable by airway morphology.