2-Nitrotoluene (2NT), but not 3-nitrotoluene (3NT) or 4-nitrotoluene (4NT), is genotoxic in the in vivo-in vitro hepatic DNA repair assay. These differences in genotoxicity may be due to hepatic metabolism. For this reason, the metabolism of the nitrotoluenes was compared in hepatocytes isolated from male Fischer 344 rats. Hepatocytes were incubated with [U-14C]2NT, 3NT, or 4NT at concentrations from 25 to 1000 microM for up to 60 min. Metabolites were separated by reverse phase HPLC and identified by coelution with standards on HPLC, specific enzyme hydrolysis, and GC-MS analysis. 2NT was converted to 2-nitrobenzyl alcohol (52%), 2-nitrobenzyl alcohol glucuronide (28%), an unidentified metabolite (20%), and 2-nitrobenzoic acid (3%). Metabolites from 3NT were 3-nitrobenzoic acid (56%), 3-nitrobenzyl alcohol (29%), and 3-nitrobenzyl alcohol glucuronide (13%). 4NT was metabolized to S-(4-nitrobenzyl)glutathione (68%), 4-nitrobenzyl alcohol (12%), sulfate and glucuronide conjugates of 4-nitrobenzyl alcohol (6%), and 4-nitrobenzoic acid (2%) (expressed as percentage of total metabolism). The formation of the respective nitrobenzyl alcohols by hepatocytes was linear with respect to time for 15-20 min. The formation of other metabolites was linear over a 45-min incubation period. Incubation of 2NT, 3NT, or 4NT (1 mM) with rat hepatic microsomes produced only the respective nitrobenzyl alcohols and the rate of formation was linear for 90 min. The data suggest that each nitrotoluene isomer is metabolized to the corresponding benzyl alcohol, but that substantial differences in the metabolism of the benzyl alcohols exist.