Mouse liver tumors frequently harbor activating ras gene mutations. Downstream effector molecules of p21 Ras include Raf-1 kinase which mediates external signals via kinase signaling pathways to nuclear transcription factors including c-Fos and c-Jun. Mouse liver tumors with differing ras-mutational status were analyzed for alterations in Ras/Raf-1 signal transduction. Tumors were characterized with respect to the presence of base substitutions in the 3 known hot-spot positions at codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras. Ha-ras codon 61 or Ki-ras codon 13 mutations, but no N-ras mutations, were detected in 23 out of 33 tumors analyzed, while no ras-mutations were found in 10 of the tumors. There was no significant difference in the expression of p21 Ras proteins between ras-mutated tumors and tumors without detectable ras mutations. To allow for determination of Raf-1 kinase activity in tumors, a sensitive and specific assay was developed for measurements with tissue homogenates. Raf-1 kinase activity was increased about four-fold in liver tumors as compared with normal liver tissue. No significant differences in kinase activity, however, were evident between ras-mutated and ras-wild-type tumors. The same was true with respect to the levels of c-fos and c-jun mRNAs. Moreover, there were no significant differences in cell division (5-bromo-2Ј-deoxyuridine-labeling indices) of hepatocytes from ras-mutated and ras-wild-type tumors. The similar degree of constitutive activation of the Ras/Raf-1 signaling pathway in liver tumors, with and without detectable ras mutations, suggests that other molecules within the signaling pathway may substitute for ras-mutations during oncogenic conversion of ras-wild-type hepatocytes. (HEPA-TOLOGY 1998;27:1081-1088.)Chemically induced hepatocarcinogenesis in rodents is a very useful tool to study critical alterations occurring during the development of normal cells into tumor cells. Precancerous lesions, which may ultimately progress into benign and malignant tumors, can be observed very early during this process. Neoplasias developing in the mouse liver are characterized by a high frequency of activating mutations in protooncogenes of the ras family that encode for small membrane-bound proteins of 21 kd. The frequency at which ras mutations occur in mouse liver tumors varies amongst different strains of mice and may exceed 50% depending on strain and treatment conditions of mice (for a recent review see Maronpot 1 ). Activating point mutations in ras-genes are almost exclusively found at only three codons, namely 12, 13, and 61. The observation that ras mutations can be detected at a very early stage during mouse hepatocarcinogenesis strongly indicates that they represent initial genetic alterations that confer a selective advantage to the mutated cells which drives their clonal expansion. [2][3][4] During the last couple of years, the role of p21 Ras proteins as part of signal transduction pathways that mediate signals from transmembrane receptors into the nucleus has b...