Nkx2.1 (TTF-1), a homeodomain-containing transcription factor essential for specific gene expression in thyroid follicular cells, exists also in the thyroidal C cells that secrete calcitonin (CT). In this report, we examined the effect of Nkx2.1 on the CT gene transcription. Luciferase reporter assay using the 2kbp promoter sequence of rat CT/CGRP gene revealed that Nkx2.1 induced a significant increase in the promoter transcription. Furthermore, we detected Pax1 and/or Pax9 gene expression in mammalian medullary thyroid carcinoma cell lines, rat rMTC and human TT cells, and in mammalian thyroid glands by RT-PCR. The Pax9 mRNA, expressed in the TT cells and rat thyroid, was then isolated by cDNA cloning. Sequence analysis showed that both rat and human Pax9 proteins contained characteristic domains: i.e. the paired domain and octapeptide motif. Alternative transcripts encoding Pax9 isoforms were not identified in the rat thyroid or TT cells. Dual luciferase assay indicated that Pax9 did not increase transcription from the CT/CGRP promoter. Pax9 also showed no cooperative effects when it was co-transfected with Nkx2.1. The results suggest that CT gene expression could be directly activated by Nkx2.1, whereas Pax9 is not involved in transcription from the 2kbp CT promoter.