In mammalian cells, nine conserved isoforms of the Na ϩ /H ϩ exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1-5 are localized to the plasma membrane, whereas NHE6 -9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ϳ15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrindependent endocytosis of transferrin via pH regulation. pH regulation; endosome; exocytosis IN MAMMALIAN CELLS, endocytosis and exocytosis are important for various intracellular events such as receptor-mediated ligand uptake and signal transduction via the receptors. The ligands and receptors are internalized through vesicles that form from the plasma membrane, migrate to endosomes, and are sorted in recycling pathways for relocation to the plasma membrane or to late endosomes/lysosomes for degradation. The lumina of endocytotic membrane compartments are gradually acidified as they are trafficked through the pathways, and the regulation of this acidification has been suggested to be important for these trafficking events (21). This acidification is primarily achieved by proton pumping into lumens by vacuolar H ϩ -ATPase (V-ATPase) (9). Recent studies suggest that luminal pH is regulated by a counterbalance between the influx of protons mediated by V-ATPase and proton leakage from the lumen through ion transport proteins such as anion channels, ClϪ /H ϩ exchangers, and Na ϩ /H ϩ exchangers (NHE) (5,15,26,37).For mammals, nine conserved NHEs (NHE1-9, also referred to as SLC9A1-9) have been identified (31). NHA1-2 (SLC9B1-2) and SLC9C1-2 have also been identified as SLC9 members, but their primary structures are distantly related to SLC9A1-9 (3, 18) (www.bioparadigms.org/slc/pdf/SLC09_2010 -09-06.pdf). In NHE1-9, five of those isoforms ...