The cDNA for TTF-1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF-1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF-1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK-2 homeodomain. TTF-1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF-1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF-1 gene is not located within previously described clusters of homeobox-containing genes.