In this paper we examine the role of thyroid hormone in regulating expression of the Na+/H+ exchanger. Thyroid hormone has been reported to regulate the activity of the Na+/H+ exchanger messenger RNA in some cell types. Treatment of cardiac myocytes with 3,5',3'-triiodothyronine results in an increased expression of Na+/H+ exchanger protein. Also, compared with euthyroid animals, hypothyroid rats express decreased amounts of the Na+/H+ exchanger protein. To examine the mechanisms involved in regulating expression of the Na+/H+ exchanger, we have characterized the regulation of a distal element of the NHE1 promoter by the thyroid hormone receptor. We have previously shown that a -1085/-800 nucleotide (nt) region of the promoter is a modular element with a -841/-800 nt activating element. Using electrophoretic mobility shift assay, we show that this element interacts with thyroid hormone receptor TRalpha(1), a nuclear hormone receptor. The addition of exogenous TRalpha increased transcriptional activity of the -841/-800 nt element of the Na+/H+ exchanger promoter. We show that TRalpha binds to a region on the -841/-800 nt element that is near, but not identical, to the previously identified chicken ovalbumin upstream promoter transcription factor-binding site. Our results are the first demonstration that thyroid hormone and the thyroid hormone receptor TRalpha(1) regulate expression of the Na+/H+ exchanger.