Four isoforms of the Na؉ /H ؉ exchanger (NHE6 -NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid-to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.The luminal ionic composition of intracellular compartments differs from the cytoplasm, and each compartment is characterized by a unique, organelle-specific ion concentration. This specific ionic composition is thought to be an important determinant for organelle function and is maintained by the concerted action of ion transport carriers on the membrane (1, 2). Organelles of the secretory and endocytic pathways exhibit differential weak acidity in their lumen with a gradient of pH values decreasing toward the trafficking destination, from ER 1 (pH ϳ7.1) to Golgi (pH ϳ6.2-7.0), trans-Golgi network (TGN) (pH ϳ6.0), and secretory granules (pH ϳ5.0) and from early and late endosomes (pH ϳ6.5) to lysosomes (pH ϳ4.5) (1, 3, 4). This progressive acidification is essential for compartmentalizing cellular events, such as post-translational modifications, sorting of newly synthesized proteins into the secretory pathway, and the degradation or recycling of internalized ligandreceptor complexes and fluid-phase solutes in the endocytic pathway (3, 5). Even pH differences of less than 0.5 between organelles can be essential for the compartmentalizing cellular events (6).The differential ionic milieu of the organelles is maintained by a suite of ion carriers on the membrane, including pumps, channels, and transporters. Luminal acidity is primarily generated by the vacuolar-type H ϩ -translocating ATPase (VATPase) (4, 5).