We have earlier shown that expression of the human activated Ki-ras, directed by the rat thyroglobulin (TG) promoter in the thyroid gland of transgenic mice, is able to induce thyroid benign tumors, albeit at low incidence. A likely explantation of our results is that the low levels of exogenous Ki-ras transcripts are not sufficient to induce multifocal tumors in the thyroid gland. We have performed experiments to analyze the effects of a similar construct in vitro upon thyroid-cell proliferation and differentiation. Transfection of FRTL-5 rat thyroid cells with the human Ki-rasval12 fused to the rat TG promoter is rapidly followed by reduced expression of the differentiation markers thyroglobulin, thyroperoxydase and thyrotropin receptor, but not by fully malignant cell transformation. The data reported support the hypothesis that Ki-ras mRNA levels are critical to the process of complete neoplastic transformation of thyroid epithelial differentiated cells in vitro.