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Basal and thyroid hormone-regulated expression of constructs containing varying lengths of 5' flanking sequence of the human glycoprotein hormone subunit gene ligated to the reporter gene encoding chloramphenicol acetyl transferase (CAT) were examined in transfected pituitary GH3 cells. CAT gene expression was quantified by measurement of CAT enzyme activity. In addition, an RNase protection assay was used to confirm changes in CAT mRNA levels and to verify regulation of transcription from the authentic gene start site. The pattern of basal promoter activity indicated the presence of inhibitory DNA elements between –98 and –172 and upstream of –846 relative to the transcriptional start site, as well as a stimulatory element between –346 and –846. Each of the transfected constructs exhibited tri-iodothyronine inhibition of promoter activity, suggesting the presence of an inhibitory thyroid hormone response element within 98 bp of the gene transcriptional start site.