The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.