DNA binding and adduct formation of aflatoxin B1 (AFB1) was studied in cultured bladder and tracheobronchial explants from human, monkey, dog, hamster and rat. Explants were exposed to (3H)AFB1 (1 micrometer final concentration) in PFHR-4 medium (pH 7.4) without serum for 24 h, after which epithelial cell DNA was isolated by hydroxylapatite chromatography. These binding levels were not correlated with the relative susceptibilities of these species to AFB1 hepatocarcinogenesis, in that the hamster and the dog are insensitive, but exhibited the highest binding, while the susceptible species, the rat and the monkey, had lower binding. After acid hydrolysis of the isolated DNA, the (3H)AFB1-DNA adducts were separated by high-pressure liquid chromatography. In some cases small amounts (0-8%) of unknown, polar adducts could be detected.