Aflatoxin B1 (AFB1) DNA binding, adduct formation, and AFB1-DNA adduct repair were studied in tracheal explants from rabbit, hamster, and rat. These species vary in populations of cytochrome P-450-containing nonciliated tracheal epithelial cells. Explants were cultured in media containing 0.5 microM AFB1 for 12 h. After the 12-h treatment, the explants were cultured for time intervals up to 84 h and then analyzed for AFB1-DNA adducts. Binding of AFB1 to DNA was highest in rabbit tracheal explants (78 pmol/mg DNA), followed by the hamster (28 pmol/mg DNA), with the rat (3 pmol/mg DNA) showing minimal AFB1-DNA binding. Repair rates in the hamster and rat were constant over time with removal of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 accounting for the majority of adduct disappearance. The rabbit demonstrated biphasic repair of adducts; all adduct types [8,9-dihydro-8-(2-amino-6-formamido-4-oxo-3,4-dihydropyrimid-5- ylamino)-9- hydroxyaflatoxin B1] were rapidly removed during the first 12 h posttreatment with AFB1, followed by a slower removal phase of primarily 8,9-dihydro-8-N7-guanyl)-9-hydroxyaflatoxin B1. After 84 h, 90, 72, and 55% of the initial adducts were removed in the rabbit, hamster, and rat, respectively. Labeled thymidine studies showed that cells of the tracheal epithelium did not turn over sufficiently to bias the apparent repair rates. These results demonstrated that carcinogen activation and repair capabilities of tracheal epithelium vary among species and that these processes likely relate to the presence of smooth endoplasmic reticulum containing nonciliated tracheal epithelial cells in those species.