DNA from B6C3F1 mouse and Fischer 344 rat liver tumors induced by N-nitrosodiethylamine (DEN) were examined for the ability to induce morphological transformation of NIH3T3 cells. DNAs from 14 of 33 of the mouse liver tumors induced by a single injection of DEN at 12 or 15 days of age were positive in this assay while DNA from only one of 28 DEN-induced rat liver tumors was active. Southern blot analysis of the NIH3T3 transformants derived from the mouse liver tumors revealed amplified and/or rearranged restriction fragments homologous to the H-ras proto-oncogene. DNA from two independent foci induced by the rat tumor DNA did not hybridize to probes for members of the ras gene family or c-raf. Activating mutations in the H-ras genes from the DEN-induced mouse liver tumors were characterized by selective oligonucleotide hybridization and the detection of a new XbaI restriction site by Southern blot analysis. In activated H-ras genes from the DEN-induced mouse liver tumor DNA, seven of 14 had a CG----AT transversion at the first base of the 61st codon, three of 14 had an AT----GC transition and four of 14 had the AT----TA transversion at the second base of codon 61. This spectrum of mutations is very similar to that recently observed in activated H-ras genes found in spontaneously occurring B6C3F1 mouse liver tumors. Taken together, the data suggest that the DEN-induced rat and mouse liver carcinogenesis may involve genetic targets other than or in addition to the H-ras gene.