By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.