Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) strongly inhibited the N-pyridine oxidation and alpha-carbon hydroxylation pathways of the in vitro metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by Syrian golden hamster liver microsomes from saline-injected (noninduced) animals. PEITC was more potent than BITC. NNK-reduction was enhanced by both isothiocyanates. Both N-oxidation and alpha-hydroxylation activities were several-fold greater in hamster liver microsomes compared with F344 rat liver microsomes. Consequently, the inhibitory effect of BITC and PEITC on rat liver microsomes (noninduced) was not as pronounced. NNK-reduction in rat liver microsomes was not significantly different from hamster and was not enhanced by BITC and PEITC. Neither BITC nor PEITC had a strong inhibitory effect on the in vitro metabolism of benzo[a]pyrene (BaP) by either hamster or F344 rat liver microsomes from beta-naphthoflavone treated animals. The extent of BaP metabolism was similar for the two microsome groups. Since the metabolism of NNK and BaP depends upon cytochrome P450-mediated reactions that may utilize different isozymes of cytochrome P450, our data suggest that BITC and PEITC may inhibit the activity of some isozymes and not others. Our results also indicate that the inhibition of the metabolism of NNK by isothiocyanates as previously described for the mouse and rat can now be extended to include the hamster as well.