The endostyle is a special organ in the pharynx of urochordates, cephalochordates and cyclostomes. During evolution of the primitive chordates, the endostyle was organized in their common ancestor(s) with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has an iodine-concentrating activity and is therefore thought to be functionally homologous to the vertebrate thyroid gland. Human TITF1 and mouse TITF1 are members of the Nkx-2.1/TTF-1 gene subfamily, which encode an NK-2 type homeodomain transcription factor. The genes are expressed in the thyroid gland and are essential for thyroid-specific structural gene expression. In the present study, we isolated cDNA clones for ascidian homologs of TITF1 from Halocynthia roretzi and Ciona intestinalis, and examined whether the genes are expressed in the ascidian endostyle. Results clearly indicated that both the H. roretzi homolog Hrtitf1 and the C. intestinalis homolog Cititf1 are expressed specifically in the endostyle. The present finding therefore provide molecular evidence for the functional relationship between the ascidian endostyle and vertebrate thyroid gland. However, the genes are expressed in the supporting element regions but not in the putative iodine-concentrating regions of the endostyle.