A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purified. It binds to the rat thyroglobulin promoter at three different sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.