The effect of phenethyl isothiocyanate (PEITC) on the metabolism of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) by cultured rat oral tissue was investigated. Two protocols were used. In one, oral tissue from untreated rats was cultured in the presence of 10 or 50 μM PEITC and either NNN or NNK. The levels of NNN and NNK metabolites released into the culture media were determined by HPLC analysis. The presence of 10 μM PEITC inhibited the formation of all NNN metabolites from 45 to 70% when the concentration of NNN was 1 μM or 10 μM. When the concentration of PEITC was 50 μM the extent of inhibition was from 70 to 90%. α-Hydroxylation of NNK was inhibited 70 to 90% and N-oxidation of NNK was inhibited 80 to 90% by 10 μM PEITC. Carbonyl reduction of NNK to NNAL was unaffected by 10 μM PEITC and only slightly inhibited by 50 μM PEITC. In the second protocol, rats were fed NIH-07 diet containing 3 μmol PEITC/g for 1-14 days. The metabolism of NNN by cultured oral tissue from these rats was decreased from 40 to 90% relative to that by tissue from control rats. NNK metabolism was inhibited 40 to 60%. The extent of inhibition was the same when rats were fed PEITC containing dlet for 1 or 14 days. NNN and NNK are the only tobacco constituents which induce oral cavity cancer in an animal model. The results of this study suggest the possibility that PEITC may be useful as a chemopreventive agent for oral cavity cancer.