The persistence of pyridyloxobutyl DNA adducts in lung and liver of F-344 rats treated with the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was investigated. The levels of these adducts were determined at various time points up to 4 weeks post s.c. injection of [5-3H]NNK (0.8 mg/kg body wt). Maximal levels of the adducts were observed between 4 and 24 h in both tissues. The disappearance of the adducts from lung and liver DNA was multiphasic with initial half-lives of 50 and 38 h respectively. In both cases, detectable levels of the pyridyloxobutyl adducts were observed at 4 weeks post injection. The in vitro rate of adduct disappearance was studied using calf thymus DNA reacted with 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone in the presence of esterase. Adduct levels were measured for up to 2 weeks after the initiation of the experiment. The decomposition of these adducts was triphasic with half-lives of 6, 120 and 430 h. The multiphasic disappearance of the pyridyloxobutyl adducts suggests that there is more than a single adduct generated upon pyridyloxobutylation of DNA and that at least one of these adducts has a significant lifetime in DNA.