Activating mutations in RAS proto-oncogenes are associated with several different histotypes of thyroid cancer, including anaplastic thyroid carcinoma. The latter is the most aggressive cancer of the thyroid gland, showing little or no expression of the differentiated phenotype. Likewise, expression of viral RAS oncogenes in FRTL-5 rat thyroid cells mimics such loss of differentiation. We established FRTL-5 cell lines stably expressing constitutively active forms of RAS, either of viral (v-Ha-RAS or v-Ki-RAS) or cellular (H-RAS^V12) origin and generated a tamoxifen-inducible RAS oncoprotein to analyze the timing of RAS effects on thyroid differentiation. In RAS-transformed FRTL-5 cells we measured the expression of many thyroid-specific genes by real-time PCR, and observed that a clear loss of differentiation was only obtained in the presence of high RAS oncogene expression. In contrast, thyrotropin-independent growth appeared to be induced in teh presence of both low and high levels of oncogenic RAS expression. We also showed that inhibition of differentiation is an early RAS-induced phenomenon. Finally, we demonstrated that only high doses of RAS oncogenes are able to inhibit the activity of Titf1 and Pax8, two transcription factors essential for the maintenance of thyroid differentiation, and that the homeodomain of Titf1 is a target of the inhibitory action of RAS. Our results represent the first evidence of a dose-dependent effect of RAS oncogenes on thyroid epithelial differentiation.