The liver tumor-prone B6C3F1 mouse (C57Bl/6 female x C3H/He male), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB-induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB-induced tumors but in only one of five spontaneous tumors, whereas the level of Ha-ras mRNA increased in nine of 10 PB-induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors.