Dinitrotoluene (DNT), an important industrial nitroaromatic compound, is a potent hepatocarcinogen in rats. Male Fischer-334 rats fed technical-grade DNT in the diet for 12 months showed a 100% incidence of hepatocellular carcinomas1. However, various in vitro and in vivo short-term tests using several genotoxic end points have failed to show activity with DNT or purified DNT isomers2–6, including unscheduled DNA synthesis (UDS) in primary hepatocytes in vitro7. In the in vivo–in vitro hepatocyte DNA repair assay, which measures chemically induced UDS in hepatocytes isolated from rats treated in vivo8, DNT was found to be a potent UDS inducer9 having a peak of activity 12 h after a single dose. A possible explanation for the discrepancy between the in vivo and in vitro results is that metabolism by gut flora may be required for formation of the proximate or ultimate carcinogenic metabolites of DNT. The importance of biotransformation of toxicants by intestinal microflora has been demonstrated for cycasin10,11 and other xenobiotics12. We have used the in vivo–in vitro hepatocyte DNA repair assay to study the role of intestinal microflora in DNT genotoxicity. We report here extensive DNT-induced DNA repair in rats having the normal complement of gut flora, but not in rats which have no gut flora. These results indicate that metabolism of DNT by gut flora is a necessary step in the genotoxicity of this compound and illustrate the value of the in vivo–in vitro hepatocyte DNA repair assay in assessing the carcinogenic potential of nitroaromatic compounds.