The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. In order to identify such determinants in the gastric H,K-ATPase, fusion proteins of YFP and the gastric H,K-ATPase -subunit (YFP-) and CFP and the gastric H,K-ATPase -subunit (CFP-) were expressed in HEK-293 cells. Then plasma membrane delivery of wild type CFP-, wild type YFP- and YFP- mutants lacking one or two of the seven -subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild type YFP- resulted in the plasma membrane localization of the protein, while the expressed CFP- was retained intracellularly. When co-expressed, both CFP- and YFP- were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP- prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the -subunit are essential for the plasma membrane delivery of the -subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the -subunits from different species, is not critical for the plasma delivery of the protein.