AbstractWe have examined the mechanism of activation of c‐raf‐1 and the expression of epidermal growth factor receptor (EGF‐R) gene in the three relatively radiation resistant human squamous cell carcinoma‐derived cell lines (SCC‐35, JSQ‐3, and SQ‐20B). The human c‐raf‐1 sequences were observed in the primary G418r, human Alu+NIH/3T3 clones transfected with these tumor cell DNAs. The humanraf‐1‐related sequences were truncated, rearranged, and amplified in the primary NIH/3T3 transfectants, and a majority of the clones revealed sequences corresponding to the carboxy‐terminus of the protein product of humanraf‐1 (Raf‐1). A highly tumorigenic potential of transfectant clones correlated with expression of the new (human)raf‐1‐related sequences. By contrast, the EGF‐R gene was amplified and aberrantly overexpressed in only the SQ‐20B tumor cells. The human EGF‐R‐related sequences were not detected in the human Alu+NIH/3T3 transfectant clones, including those derived following the SQ‐20B tumor cell DNA transfection. The levels of Raf‐1‐associated serine/threonine kinase activity were comparable in SCC‐35 and SQ‐20B cells. We have previously reported that the antisense c‐raf‐1 cDNA transfection is sufficient for the down regulation of tumorigenicity and radioresistance in SQ‐20B cells. The present data suggest that the role of c‐raf‐1 in the development of the tumor phenotype may be unrelated to the amplification and activation of EGF‐R in t