The metabolism of radiolabeled dinitrobenzene (DNB) isomers was compared in hepatocytes and hepatic subcellular fractions isolated from male Fischer-344 rats. Under aerobic conditions, reduction was the major metabolic pathway for m- and p- DNB in hepatocytes with m- and p-nitroaniline accounting for 74.0 +/- 1.2 and 81.0 +/- 0.6% (mean +/- SE, n = 4), respectively, of the radioactivity present after a 30-min incubation. The major metabolite of o-DNB in similar incubations was S-(2-nitrophenyl)glutathione which represented 48.1 +/- 5.5% of the total radioactivity; o-nitroaniline accounted for 29.5 +/- 2.1% of the radioactivity. Incubations of DNBs with microsomes produced nitroanilines as well as nitrosonitrobenzenes and nitrophenylhydroxylamines. Reduction of o- and m-DNB by microsomes was NADPH-dependent. Reduction of p-DNB could be supported by NADH as well as NADPH, although the rate of reduction was slower with NADH. o- and p-DNB were reduced to nitroanilines 3-5 times faster than m-DNB in hepatocyte and microsomal incubations. Conjugation of o- and p-DNB, but not m-DNB, with glutathione occurred in cytosol incubations although only o-DNB formed the glutathione conjugate in intact hepatocytes. Thus, there are substantial isomeric differences in the aerobic metabolism of the DNBs by rat hepatic enzymes.