Constitutive activation of the RAS-RAF-MEK-ERK signaling cascade plays an important role in the pathogenesis of AML. We were interested whether abnormalities of the serine-threonine kinases A -, B, - and C -RAF occur in AML and whether they might be causative for activation of this pathway. Expression of these genes was assessed by real-time quantitative (rtq) PCR in 102 patient samples of AML, three AML cell lines and normal CD34+ cells of seven individuals. Results were correlated with the phosphorylation status of ERK 1/2, as measured by immunoblot analysis. 45/82 (54,9%) of AML cases showed ERK phosphorylation indicating constitutive activation of the pathway. However, there was no correlation with expression levels of either A-, B- or C-RAF. Deletions of the B-RAF gene as revealed by rtq PCR were observed in 20/99 (20%) of AML cases and were consistent with cytogenetic results but did not correlate with ERK activity, either. In addition, we screened for mutations of B- and C-RAF in all samples showing ERK phosphorylation using PCR and sequence analysis. Two novel C-RAF missense mutations, both of them located in the kinase domain, were detected in the subgroup of therapy related AML (t-AML): S427G in one and I448V in another patient. Expression of the mutated proteins in mammalian cells resulted in the constitutive activation of MEK and ERK, thus strongly suggesting that the mutations activated C-RAF. Sequence analysis of the primary tumors (colon cancer and teratoma, respectively) as well as buccal epithelial cells revealed these mutations of germline origin and denaturing high pressure liquid chromatography revealed a frequency of <1/400 alleles in the healthy population. Immunohistochemistry using antibodies against phosphorylated ERK demonstrated the pathway to be constitutively activated in the primary tumors as well but inactive in non-neoplastic tissue. In conclusion, we have identified the first activating missense mutations of the C-RAF proto-oncogene in a human tumor. Since both are of germline origin and the primary as well as secondary malignancies showed constitutive activation of the RAS-RAF-MEK-ERK pathway, these mutations might be regarded as predisposing factors in these individuals.