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.