The renal type II Na-Pi cotransport is the rate-limiting step in proximal tubular phosphate (Pi) reabsorption. Among the different "proximal tubular" cell lines, this transporter seem only to be expressed in opossum kidney cells (OK cells). We have isolated the 5'-flanking region of the ok-Npt2 gene (OK cell type II Na-Pi cotransporter) including exons 1-3 and containing a TFIID site (TATA box), a GCCAAT site, an AP1 site, and two microsatellite GGAA repeats. Major transcription initiation sites were determined by primer extension and rapid amplification of 5' cDNA ends (5'-RACE). A 327-bp fragment containing the TFIID and GCAAT element was driving the downstream luciferase reporter gene in homologous transfection assays. Slightly reduced promoter activity was observed with a 198-bp fragment containing the GCAAT element; shorter fragments were without activity. Promoter activity (327-bp fragment) could also be observed in transfections into HeLa cells but not in U937 human macrophage cells, MCT mouse kidney cortex cells, and MDCK cells. Different "physiological" stimuli known to be associated with altered proximal tubular Na-Pi cotransport activity are without effect on transcriptional activity in above homologous transfection experiments.