Species- and strain-specific spontaneously occurring tumors have been observed in rodents maintained under normal laboratory conditions. Elucidation of the molecular mechanisms associated with the development of these spontaneous tumors may provide a better understanding of tumor development associated with exposure to chemical carcinogens. In view of the high frequencies of oncogene activation shown in rodent tumors induced by known chemical carcinogens, we have investigated oncogene activation in spontaneous tumors of the B6C3F1 mouse and Fischer 344/N rat by DNA transfection techniques. A marked difference in the presence of activated oncogenes in spontaneous rat tumors versus spontaneous mouse liver tumors was observed in this study. All rat tumors tested failed to yield activated oncogenes (0/29), whereas 30% (3/10) of mouse hepatocellular adenomas and 77% (10/13) of hepatocellular carcinomas scored positive by DNA transfection. These transforming genes were identified as an activated Ha-ras gene in all the adenoma transfectants and in 8 of the 10 carcinoma transfectants. The two remaining hepatocellular carcinomas contained transforming genes that appear not to be members of the known ras gene family. The B6C3F1 mouse liver system might provide a very sensitive assay not only for assessing the potential of a chemical to activate a cellular proto-oncogene, but also for detecting various classes of proto-oncogenes that are susceptible to mutational activation.