The sodium-phosphate co-transporter 2a (NPT2a) is the principle phosphate transporter expressed in the brush-border of renal proximal tubules and is down regulated by parathyroid hormone (PTH) through an endocytic mechanism. Apical membrane expression of NPT2a is dependent upon interactions with the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1). An LLC-PK1 renal cell line stably expressing the PTH receptor (PTH1R) and NHERF-1, termed B28-N1, fails to functionally express NPT2a. In B28-N1 cells, NHERF-1 and NPT2a are inappropriately localized to the cytoplasm. Ezrin, in the activated state, is capable a linking NHERF-1-assembled complexes to the actin cytoskeleton. Early passage LLC-PK1 cells stably transfected with either empty vector or wild type ezrin express a comparable level of the active, T567 phosphorylated form of ezrin and are capable of functionally expressing NPT2a. Co-localization of the PTH1R, NPT2a and ezrin exists and are prominently associated with actin-containing microvilli in apical domains of these cells. Upon PTH treatment, the PTH1R, NPT2a, NHERF-1 and ezrin co-localize to endocytic vesicles and NPT2a-dependent phosphate uptake is markedly inhibited. LLC-PK1 cells expressing the constitutively active ezrin (T567D) display enhanced NPT2a functional expression and PTH-mediated regulation of phosphate. Expression of a dominant negative ezrin, consisting of the N-terminal half of the protein, markedly disrupts NPT2a-dependent phosphate uptake. PTH does not appear to alter ezrin phosphorylation at T567. Instead, PTH perhaps initiates NPT2a endocytosis by inducing re-organization of the actin-containing microvilli in a process that is blocked by the actin-stabilizing compound, jasplakinolide.