The NHE6 protein is a unique Na+/H+ exchanger (NHE) isoform, believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively-charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. (i) When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. (ii) When green fluorescent protein (GFP) was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the ER membrane. The localization pattern was the same as that of fusion protein in which GFP was fused after H2 of NHE1. (iii) In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. (iv) Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.