Sodium-hydrogen exchanger regulatory factor (NHERF-1) was initially identified as a renal proximal tubule brush border membrane (BBM) adaptor protein that binds to NHE3 (sodium-hydrogen exchanger 3) and ezrin thereby facilitating the formation of a macromolecular signal complex that promotes cAMP-dependent protein kinase (PKA) phosphorylation and inhibition of NHE3 activity. A second member of this family, NHERF-2, has also been cloned. Although sharing a similar modular structure including two tandem highly conserved PDZ (PSD-95/Dlg/ZO-1) protein interaction domains and an ezrin-radixin-moesin-merlin binding domain, NHERF-1 and NHERF-2 are different gene products, have overlapping but not identical protein targets, and may mediate different cell functions. Rat renal proximal tubules express only NHERF-1 whereas human and mouse proximal tubules express both isoforms. Although both NHERF-1 and NHERF-2 have the capacity to bind the apical membrane transporters NHE3 and the sodium-dependent phosphate co-transporter IIa (Npt2) using in vitro assays, the precise role of each of the NHERF isoforms in intact tissue is unknown. To define the physiological role for NHERF in the kidney and to differentiate the role of NHERF-1 from NHERF-2, we generated an NHERF-1 null mouse line using homologous recombination techniques. In NHERF-1 (-/-) mice, NHERF-1 was absent from all tissue but the expression and cellular location of NHERF-2 was only minimally different from wild-type animals. Serum electrolytes were normal in the NHERF-1 (-/-) mice except for a modest decrease in the serum phosphate concentration. The glomerular filtration rate and systemic arterial blood pressure was not different from wild-type mice. The morphology of the kidney of the null mice was also normal and the distribution of actin and ezrin was similar to wild-type animals. In wild-type animals, NHERF-1 was expressed predominantly in the brush border membrane (BBM) while NHEFR-2 was expressed at the base of the microvillus membrane. In wild-type animals, however, some co-localization of the two NHERF isoforms was identified using an array of antibodies. Immunoprecipitation experiments in wild-type mice indicated that NHERF-1 and NHERF-2 formed heterodimers, and that each isoforms co-immunopreciptitated NHE3 and Npt2. In null mice, NHE3 was appropriately targeted to BBM of renal proximal tubule cells and BBM abundance of NHE3 was the same as in wild-type animals. Immunoprecipitation of NHERF-2 from the kidney of null animals resulted in recovery of NHE3. Nonetheless, as compared to wild-type animals, there was a significant deficit in cAMP-associated inhibition of NHE3 activity as assayed in isolated BBM from NHERF-1 (-/-) mice despite the presence of functionally active PKA. This clearly demonstrates that while NHERF-2 showed equivalent activity as NHERF-1 in cAMP-mediated inhibition of NHE3 in model PS120 fibroblasts, NHERF-2 could not substitute for the loss of NHERF-1 in the kidney of mutant mice. As compared to wild-type controls, NHERF-1 null mice demonstrated an increase in the urinary excretion of phosphate while on a regular or low phosphate diet. BBM staining for Npt2 was decreased and BBM abundance of Npt2 was significantly less than wild-type animals. Immunoprecipitation of NHERF-2 from null mice kidneys did not result in recovery of Npt2 suggesting that in the mouse, only NHERF-1 interacts with Npt2. In NHERF-1 (-/-) mice, not only was BBM Npt2 abundance decreased, Npt2 appeared to be mistargeted to intracellular vesicular structures. Using confocal microscopy, Npt2 was demonstrated to accumulate in the Golgi complex of proximal tubule cells. These studies demonstrate a unique subcellular distribution of NHERF-1 and NHERF-2 in the proximal tubule of the mouse kidney. While each protein has the capacity to bind to the renal apical membrane transporters NHE3 and Npt2, at least in in vitro assays, only NHERF-1 mediates PKA regulated inhibition of NHE3 activity. The role of NHERF-2 is unknown but speculate that it may be required for cellular targeting of NHE3 or its retention in the apical membrane. The NHERF-1 (-/-) mice also demonstrate an increase in the urinary excretion of phosphate associated with a decrease in Npt2 expression in BBM and the accumulation of Npt2 in the Golgi. Thus, NHERF-1 but not NHERF-2 is required for the targeting and/or apical retention of Npt2 in the renal proximal tubule of the mouse. On the other hand, there appears to be little direct interaction between Npt2 and NHERF-2 proteins. Since their initial description, NHERF-1 and NHERF-1 have been found to interact with 30 or more target proteins including other transport proteins and ion channels in addition to NHE3 and Npt2, hormone receptors, signalling proteins, structural elements, and transcription factors. The NHERF-1 (-/-) mouse and the subsequent development of the NHERF-2 (-/-) mouse should prove to be valuable models for study of the integrated function of PDZ proteins in the kidney.