The effect of phenethyl isothiocyanate (PEITC), a dietary inhibitor of carcinogenesis, on the metabolism of the tobacco specific nitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) by cultured rat peripheral lung tissues was investigated. Initially, the metabolism of NNK by the tissues was studied by incubating the lung explants in medium containing 1 and 10 μM [5-3H]NNK for 3, 6, 12, and 24 h. NNK metabolites were analyzed and quantified by HPLC and expressed as nmol/mg DNA. NNK was metabolized by three pathways; alpha-carbon hydroxylation, pyridine N-oxidation and carbonyl reduction. The principal metabolic pathway involved the conversion of NNK to the pyridine N-oxidized metabolites: 4(methylnitrosamino)-l-(3-pyridyl-N-oxide)-l-butanone (NNK-Akrade) and 4-(methylnitrosamino)-l-(3-pyridyl-N-oxide)-l-butanol (NNAL-N-oxide). When combined, NNK-N-oxide and NNAL-N-oxide constituted - 70% of the total metabolites in the medium at 24 h. To determine the effects of PEITC on the metabolism of NNK, lung explants were either treated with both 10 μM [5-3H]NNK and PEITC 10, 50, and 100 μM) for 24 h, or they were pre-treated with these same concentrations of PEITC for 16 h and then co-treated with both PEITC and 10 μM [5-3H]NNK for 24 h. In both treatment series, PEITC inhibited the alpha-carbon hydroxylation and pyridine N-oxidation of NNK and 4-(methylnitrosamino)-l-(3-pyridyI)-l-butanoI (NNAL), which is produced from NNK by carbonyl reduction. In general, the inhibition of NNK metabolism was greater when the explants were pre-treated with PEITC. These results suggest that PEITC is an effective inhibitor of the conversion of NNK to metabolites that elicit DNA damage. Our results are in agreement with previously published data in which PEITC was shown to inhibit NNK metabolism and tumorigenesis in the rat lung.