Na-phosphate (P(i)) cotransporters in the apical membrane of renal proximal tubular cells play a major role in the maintenance of P(i) homeostasis. Although two such cotransporters, Npt1 and Npt2, have been identified, little is known about the function and regulation of Npt1. We cloned and characterized the murine (Npt1) and human (NPT1) genes, isolated the 5'-flanking region of Npt1, and analyzed its promoter activity. Npt1 is approximately 29 kb with 12 exons, whereas NPT1 is approximately 49 kb with one additional exon. The Npt1 promoter has a TATA-like box but no CAAT box, and the transcription start site was identified by primer extension and 5'-rapid amplification of cDNA ends. Transfection of opossum kidney cells with Npt1 promoter-reporter gene constructs demonstrated significant activity in a 570-bp fragment that was completely inhibited by cotransfection with the transcription factor, hepatocyte nuclear factor (HNF)-3 beta. Deletion of 200 bp from the 3'-end of the 570-bp fragment abrogated its promoter activity. In addition, promoter activity of a 4.5-kb fragment, but not the 570-bp fragment, was stimulated fourfold by cotransfection with HNF-1 alpha. Other well-characterized cis-acting elements were identified in the Npt1 promoter. We suggest that Npt1 expression is transcriptionally regulated and provide a basis for the investigation of Npt1 function by targeted mutagenesis.