The ubiquitously-expressed human Na+H+ exchanger (NHE-1) contains three consensus sites (Asn-X-Ser/Thr) for N-linked glycosylation at asparagines 75, 370, and 410. The first extracellular loop is rich in serine and threonine residues which may contain O-linked carbohydrate. In order to determine unambiguously the sites of glycosylation and their role in biosynthesis and cation transport, site-directed mutagenesis at the individual potential N-glycosylation sites (Asn to Asp) was performed and all possible double and triple mutants were constructed. The mutated DNAs were expressed in PS120 hamster fibroblasts lacking endogenous exchanger, and the transfected cells were selected by their ability to survive acute intracellular acidification. All constructs produced functional exchangers that had transport rates and pharmacological profiles that were similar to that of wild-type. Immunoblot analysis of the expressed proteins with and without N-glycosidase F treatment showed that only the first N-glycosylation site (Asn 75) is utilized. In addition, treatment of NHE-1 with neuraminidase and O-glycosidase demonstrated that NHE-1 also contains O-linked oligosaccharide. Two forms of NHE-1 was consistently observed, a mature form with a molecular mass of 110,000 Da which contains N-linked and O-linked oligosaccharide and is expressed at the cell surface, and a lower molecular mass form (85,000 Da) present in the endoplasmic reticulum which only contains N-linked high-mannose oligosaccharide. NHE-3, an apically-expressed epithelial isoform which does not possess the N75 N-linked putative glycosylation site and any extracellular loops enriched in serine and threonine residues, does not exhibit any detectable glycosylation.