beta 1 and beta 2 are functional thyroid hormone receptors (TRs) that are generated from the same genomic locus by splicing of a different amino terminus onto a common carboxyl region containing the DNA and hormone binding domains. TR beta 1 is widely expressed whereas TR beta 2 is found primarily in the pituitary gland although low levels of expression have been described in other tissues. To gain insight into the mechanisms governing expression of this complex transcriptional unit, we cloned mouse genomic fragments containing the common carboxyl terminus as well as the unique TR beta 2 amino-terminal sequence that was located at least 25 kilobases upstream. The DNA and ligand binding exons are identical in size and location of their boundaries to those of the human TR beta 1 gene. To determine whether the region 5' of the TR beta 2 amino terminus represented the promoter region, we examined it for sites of transcriptional initiation and for its ability to function as a promoter in TR beta 2-expressing thyrotrope cells. Multiple transcriptional start sites extending over 400 base pairs (bp) were identified with those more proximal showing inhibition by T3. Transcription was not detected more than 400 bp upstream from the putative AUG codon, although initiation downstream of this AUG was demonstrated indicating alternative AUG usage. A fragment containing 500 bp of the TR beta 2 5'-region exhibited preferential promoter activity when transfected into thyrotrope cells that express endogenous TR beta 2. Deletion studies demonstrated that removal of consensus binding sites for the transcription factor Pit-1 resulted in loss of this cell specificity. We therefore conclude that the promoter region responsible for expression of the TR beta 2 isoform in pituitary thyrotropes is distinct from that described for TR beta 1 and is located many kilobases upstream from their common exons.