The cyclic AMP-mediated transcriptional regulation of the enzyme thyroperoxidase by thyrotropin (TSH) in thyroid follicular cells was examined at the molecular level. The 5' end of the human thyroperoxidase gene was isolated and sequenced and the transcription start site was mapped by S1 nuclease analysis. A 0.9 kilobase pair DNA fragment of the promoter was shown to confer responsiveness to thyrotropin, and cyclic AMP, in transient expression assays using two different reporter genes. Several potential sites for specific interaction with nuclear transcription factors which could be involved in the regulation of thyroperoxidase gene transcription were identified.