All three dinitrobenzene (DNB) isomers cause methemoglobinemia, but only 1,3-DNB produces testicular toxicity in rats. In order to determine whether major differences exist in the routes of DNB metabolism, male Fischer-344 rats were given an oral dose (0.15 mmol/kg) of 14C-labeled 1,2-, 1,3-, or 1,4-DNB, and excreta were collected over 48 hr. Elimination of radiolabel was rapid; 85%, 60%, and 75% of the 1,2-, 1,3-, and 1,4-DNB dose was recovered in 24 hr, respectively. Urine was the primary route of excretion, accounting for 82% of the total dose of 1,2-DNB and 75% of the dose of 1,4-DNB after 48 hr. Radiolabel from 1,3-DNB was excreted to a slightly lesser extent in the urine (63% of the dose). A greater portion of radiolabel was excreted in the feces than with the other isomers (18% of total dose, compared to 8% and 9% with 1,2-DNB and 1,4-DNB, respectively). The major urinary metabolites of 1,2-DNB were S-(2-nitrophenyl)-N-acetylcysteine (42% of the dose), 2-nitroaniline-N-glucuronide (4%), 4-amino-3-nitrophenylsulfate (17%), 2-amino-3-nitrophenylsulfate (1.5%), and 2-(N-hydroxylamino)nitrobenzene (1-2%). The major urinary metabolites of 1,3-DNB were 3-aminoacetanilide (22%), 4-acetamidophenylsulfate (6%), 1,3-diacetamidobenzene (7%), and 3-nitroaniline-N-glucuronide (4%). The major metabolites of 1,4-DNB were 2-amino-5-nitrophenylsulfate (35%), S-(4-nitrophenyl)-N-acetylcysteine (13%), and 1,4-diacetamidobenzene (7%). These results suggest that the DNB isomers are primarily metabolized by nitro group reduction and conjugation with glutathione. The testicular toxicant 1,3-DNB was apparently metabolized exclusively by reduction.