Since the transcription factor Zfhep is expressed in somatotropes and binds the rat growth hormone (rGH) gene T3-response element (TRE), we investigated whether Zfhep regulates the response of this gene to T3. In cotransfection experiments, Zfhep did not regulate the native rGH promoter in the absence of T3. However, Zfhep repressed T3-mediated activation significantly in either GH(3) or JEG-3 cells. Up to 70% repression was mediated through the rGH TRE in a heterologous promoter (thymidine kinase), but was not observed with the idealized DR4 or chicken lysozyme F2 TREs. Zfhep apparently does not repress T3-mediated activation simply by competition for binding to DNA since the C-terminal DNA-binding domain of Zfhep (which is sufficient for DNA-binding) is not sufficient for repression and since cotransfection of excess thyroid hormone receptor (TR) did not prevent repression by Zfhep. These data indicate that the rGH TRE is a composite element that can integrate Zfhep and T3 regulation.