The type IIa Na+-Pi cotransporter (NaPi-IIa) and the Na+/H+ exchanger regulatory factor-1 (NHERF1) colocalize in the apical membrane of proximal tubular cells. Both proteins interact in vitro. Herein the interaction between NaPi-IIa and NHERF1 is further documented on the basis of coimmunoprecipitation and co-pull-down assays. NaPi-IIa is endocytosed and degraded in lysosomes upon parathyroid hormone (PTH) treatment. To investigate the effect of PTH on the NaPi-IIa-NHERF1 association, we first compared the localization of both proteins after PTH treatment. In mouse proximal tubules and OK cells, NaPi-IIa was removed from the apical membrane after hormonal treatment; however, NHERF1 remained at the membrane. Moreover, PTH treatment led to degradation of NaPi-IIa without changes in the amount of NHERF1. The effect of PTH on the NaPi-IIa-NHERF1 interaction was further studied using coimmunoprecipitation. PTH treatment reduced the amount of NaPi-IIa coimmunoprecipitated with NHERF antibodies. PTH-induced internalization of NaPi-IIa requires PKA and PKC; therefore, we next analyzed whether PTH induces changes in the phosphorylation state of either partner. NHERF1 was constitutively phosphorylated. Moreover, in mouse kidney slices, PTH induced an increase in NHERF1 phosphorylation; independent activation of PKA or PKC also resulted in increased phosphorylation of NHERF1 in kidney slices. However, NaPi-IIa was not phosphorylated either basally or after exposure to PTH. Our study supports an interaction between NHERF1 and NaPi-IIa on the basis of their brush-border membrane colocalization and in vitro coimmunoprecipitation/co-pull-down assays. Furthermore, PTH weakens this interaction as evidenced by different in situ and in vivo behavior. The PTH effect takes place in the presence of increased phosphorylation of NHERF1.