Altered c-Ha-ras genes have been frequently detected in the DNA of spontaneous or chemically induced mouse liver tumors. To determine if ras gene mutation is a frequent event during liver carcinogenesis in rats, we examined the transforming activity of DNA from liver tumors that developed in rats injected with methyl(acetoxymethyl)nitrosamine (DMN-OAc) after a partial hepatectomy. Three weeks after the injection of DMN-OAc, rats were fed a diet containing phenobarbital. This carcinogen acts only on replicating liver cells. Six of eight tumor DNAs induced the transformation of NIH 3T3 cells. The transforming activity was stable upon a second round of transfection, and the transformants were tumorigenic in nude mice. Southern blot analysis of transformant DNAs showed that the transforming activity was not due to the acquisition of a ras (Ha, Ki, or N), neu, myc, A-raf, v-raf, erbA, or erbB gene of rat origin. Several transformants' restriction enzyme sensitivity was analyzed, and their activity indicated that similar transforming sequences were present in at least two tumors and that one tumor contained two different transforming sequences. These results suggest that during hepatocarcinogenesis induced in rats by DMN-OAc, alterations in the ras gene family occur infrequently or not at all and that several different genes (which are not homologous to common oncogenes) become activated and are capable of transforming NIH 3T3 cells.