2,6-Dinitrotoluene (2,6-DNT) is a potent hepatocarcinogen in Fischer-344 rats, while its 2,4-isomer is believed to be noncarcinogenic. Neither 2,6-DNT nor 2,4-DNT is carcinogenic in the strain A mouse lung tumor bioassay. To explore the possible reasons for these differences in tumor responses, we have studied the in vitro metabolism and DNA binding of 2,6-DNT in cultured hepatocytes of the Fischer-344 rat and the A/J mouse, and have also investigated the in vivo DNA binding of 2,6-DNT and 2,4-DNT in these two species. In vitro metabolism of 2,6-DNT by rat and mouse hepatocytes was similar and resulted mainly in the formation of 2,6-dinitrobenzyl alcohol, either unconjugated or as a glucuronide (57.5 to 85.5% of the total per fraction), with smaller amounts of polar, acidic metabolites (8.4 to 38.7%) and minor amounts (1.2 to 5.3%) of 2-amino-6-nitrotoluene. Anaerobic metabolism of 2,6-DNT by an extract of rat or mouse cecal contents resulted mainly in the formation of 2-amino-6-nitrotoluene and 2-(N-acetylamino)-6-nitrotoluene, and minor amounts of 2,6-diaminotoluene. Ip administration of 2,6-DNT or 2,4-DNT (150 mg/kg each) to Fischer-344 rats resulted, after 24 hr, in covalent binding to DNA of the liver (131.1 to 259.9 pmol 2,6-DNT/mg DNA; 215.4 to 226.8 pmol 2,4-DNT/mg DNA), and lower binding to DNA of the lungs and the intestine (14.9 to 22.7 pmol 2,6-DNT/mg DNA; 45.0 to 75.0 pmol 2,4-DNT/mg DNA). Similar treatment of A/J mice resulted in lower binding in the liver (25.9 to 31.9 pmol 2,6-DNT/mg DNA; 42.6 to 58.9 pmol 2,4-DNT/mg DNA), no detectable binding of 2,6-DNT in extrahepatic tissues and low amounts of binding of 2,4-DNT to lung and intestinal DNA (9.7 to 39.0 pmol/mg DNA). In vitro binding of 2,6-DNT to DNA of cultured hepatocytes from both A/J mice and Fischer-344 rats required prior metabolism of 2,6-DNT by the respective extracts from cecal contents. DNA binding was non-detectable in hepatocytes incubated with 2,6-DNT only. It is concluded that binding of 2,6-DNT to liver DNA requires its prior reductive metabolism, probably by intestinal microorganisms, and that the higher binding of 2,6-DNT in the Fischer-344 rat than in the A/J mouse may, in part, be responsible for the high susceptibility of the Fischer-344 rat to 2,6-DNT carcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)