Tunicamycin (TM) was used in toad urinary bladder (TBM) cells to study the role of N-glycosylation of the beta-subunit of Na+-K+-ATPase. Inhibition of the beta-subunit core glycosylation was dose dependent and coincided with a specific 70% decrease in newly synthesized beta- and alpha-subunits. Na+-K+-ATPase activity paralleled the decrease in the cellular content of the alpha-subunit, although the cellular and cell surface-expressed Na+-K+-ATPase pool was progressively filled up with nonglycosylated beta-subunits. In addition, the decrease in maximal Na+ transport capacity of the Na+-K+-ATPase as assessed by short-circuit current (SCC) measurements in the presence of amphotericin B correlated with the decrease in the total cell surface-expressed beta-subunit population despite the fact that it was composed of 47% nonglycosylated beta-subunits after 42 h of TM treatment. These results are consistent with the interpretation that beta-subunit glycosylation is not important either for the enzyme's intracellular sorting to the plasma membrane or its hydrolytic and transport properties. Finally, TM produced effects on basal SCC and electrical resistance that differed in their times of onset and time periods needed for recovery. Thus, in addition to the Na+-K+-ATPase, other glycoproteins in the apical membrane and the tight junctions must be implicated in the maintenance of transepithelial Na+ transport.