Hypertension is secondary to increased sodium and chloride reabsorption along the distal nephron in the kidney. The goal of this study was to characterize both structurally and functionally the recently discovered Cl-/HCO3- exchanger Pendrin (SLC26a4), which is responsible for chloride reabsorption in kidney collecting ducts. Mouse Pendrin subcloned into pTarget mammalian expression vector was stable transfected into HEK293 cells, and its kinetic parameters were determined by monitoring changes in intracellular pH (pHi) using the pH-sensitive dye BCECF. To measure the external affinity of Pendrin for Cl-, cells were first exposed to Cl- free medium and then switched to a Cl-containing medium of variable concentration, resulting in pHi decrease due to Cl-/OH- exchange. After reaching equilibrium, cells were switched back to a Cl-free medium resulting in pHi increase, and allowing determination of the internal affinity of Pendrin for Cl-. Similar experiments were performed to measure the apparent affinity of Pendrin for HCO3-. The apparent affinity constants for external Cl- and HCO3- were 1.4 and 2.8 mM respectively. Maximal Pendrin activity was higher in Cl-/ HCO3- exchange mode than in Cl-/OH- exchange mode. Pendrin was fully inhibited by DIDS (0.5mM), Diphenylamine-2- carboxylic acid (DPC) (1mM), Acetazolamide (ACZ) (1mM), and half inhibited by SITS (1mM). Structural information regarding Pendrin is limited and controversial; topological prediction programs suggest 6 different topology models with 11 to 13 transmembrane (TM) domains and 5 predicted N-glycosylation sites (N-Gs). We addressed the topology of Pendrin by checking whether predicted N-Gs and engineered N-Gs at both ends of the protein and in the regions between the putative TM segments are glycosylated. We already showed that only 2 (N167 and N172) out of the 5 predicted N-Gs are actually glycosylated, and therefore extracellular, allowing to exclude 3 of the 6 predicted topology models.