The covalent binding to hepatic RNA, DNA, and protein of a highly genotoxic dinitrotoluene (DNT) isomer (2,6‐DNT) was compared with that of a less genotoxic DNT isomer (2,4‐DNT) after oral administration to male Fischer‐344 rats. Covalent binding to each macromolecular species was proportional to dose (10 or 35 mg/kg) for each isomer, but that due to 2,6‐DNT was always 2–5‐fold higher than that due to 2,4‐DNT. There was no selectivity of either isomer for any macromolecule. The time course of appearance and disappearance of covalently bound material was similar regardless of isomer or dose administered. Little covalently bound material was present until 8 h after the dose. Covalent binding peaked between 12 and 24 h and then slowly declined. The half‐lives of covalently bound material were independent of the isomer administered, ranging from 2.9 to 5.0 d for RNA and protein and from 5.1 to 7.9 d for DNA. Both isomers disappeared from the small intestine rapidly, and covalent binding to hepatic macromolecules became significant only after the isomeric dinitrobenzyl alcohol glucuronides had appeared in the small intestine. The concentration of alcohol glucuronides in the intestine declined prior to peak covalent binding in the liver. The data suggest that covalent binding to hepatic macromolecules qualitatively reflects the differences in genotoxicities between the two isomers. The time course of intestinal disposition of the two isomers supports previous reports that suggest that activation of both isomers requires oxidation to the corresponding benzyl alcohol, glucuronidation, excretion in the bile, deconjugation, and further metabolism by intestinal microorganisms, followed by reabsorption.