In epithelial MDCK cells, the Na,K-ATPase is co-localized with adherens junctions in all stages of monolayer formation starting from initiation of cell-cell contact. The Na,K-ATPase and adherens junction proteins stay partially co-localized even after internalization due to disruption of intercellular contacts by Ca2+ deprivation. Similar to adherens junction proteins, the Na,K-ATPase is resistant to extraction with non-ionic detergent, suggesting pump association with the cytoskeleton. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase beta 1 subunit and the endogenous alpha 1 subunit is easily dissociated from the adherens junctions and cytoskeleton by detergent extraction. The MDCK cells in which half of the endogenous beta 1 subunits in the lateral membrane are substituted by unglycosylated beta 1 subunits display a slower rate of cell-to-cell contact formation and decreased ability to both spread over the surface and migrate. The lack of N-glycans in the Na,K-ATPase beta 1 subunit results in an impairment of mature cell-cell junctions as detected by an increase in the paracellular permeability of the MDCK cell monolayers and by a decrease in resistance of adherens junction proteins to extraction by a non-ionic detergent. Therefore the N-glycans of the Na,K-ATPase beta 1 subunit are important for retention of the pump at the sites of cell-cell contact. Moreover, they are important for the integrity and stability of cell-cell junctions in mature epithelia. In addition, N-glycans contribute to the formation of cell-cell contacts between surface-attached dispersed cells by mediating lamellipodia formation and stabilizing the newly formed adherens junctions.