Thyroid transcription factor I (TTF-1) plays a critical role in thyroid organogenesis and in the control of expression of several thyroid-specific genes, like those coding for thyroglobulin and thyroperoxidase. We have expressed the isolated DNA-binding homeodomain of TTF-1 in cultured thyroid cells by transient transfection. A specific reduction in the activity of co-transfected thyroglobulin and thyroperoxidase promoters was observed in the presence of the isolated TTF-1 homeodomain, as compared to their activity measured in the presence of a mutated homeodomain unable to bind DNA. The activity of the SV40 early promoter, used as a control, was only marginally affected in these experiments. The transactivation-deficient form of TTF-1 described here may thus be used for investigating other cellular processes that are dependent on TTF-1 transcriptional activity.