The rat S14 gene is synergistically regulated by thyroid hormone and carbohydrates. The 5'-flanking region of this gene contains both carbohydrate and thyroid hormone response elements (TREs). We recently located the carbohydrate response element (CHORE) between -1583 and -1069 bases from the start site of transcription that is required for glucose induction of this gene. We have now studied the relationship between the effects of CHORE, TREs, and thyroid hormone receptor on the carbohydrate regulation of transcription. We used a transient cotransfection assay with a plasmid containing 5 kilobases (kb) of the up-stream region from the S14 gene ligated to a luciferase reporter and a thyroid hormone receptor expression vector. Unliganded thyroid hormone receptor reduced the response of the reporter to glucose. Ligand-bound thyroid hormone receptor significantly enhanced the glucose response from the 5-kb promoter. While deletion of the CHORE (5-kb delta CHORE) from the 5-kb S14 promoter eliminated the glucose response without cotransfection of the thyroid receptor, deletion of the CHORE did not affect the glucose response when both thyroid hormone receptor was cotransfected and thyroid hormone was present. However, deletion of the TREs (-3261 to -2110) from the 5-kb delta CHORE construct abolished the effect. These data suggest that the thyroid hormone receptor functions as or in association with a trans-acting glucose response factor. The site of interaction between thyroid hormone and glucose is localized to an area of the gene that is different from the previously described CHORE.