Initial folding is a prerequisite for subunit assembly in oligomeric proteins. In this study, we have compared the role of co-translational modifications in the acquisition of an assembly-competent conformation of the beta subunit, the assembly of which is required for the structural and functional maturation of the catalytic Na,K-ATPase alpha subunit. Cysteine or asparagine residues implicated in disulfide bond formation or N-glycosylation, respectively, in the Xenopus beta1 subunit were eliminated by site-directed mutagenesis, and the assembly efficiency of the mutants and the functional expression of Na+,K+ pumps were studied after expression in Xenopus oocytes. Our results show that lack of each one of the two most C-terminal disulfide bonds indeed permits short term but completely abolishes long term assembly of the beta subunit. On the other hand, lack of the most N-terminal disulfide bonds allows the expression of a small number of functional Na+,K+ pumps at the cell surface. Elimination of all three but not of one or two glycosylation sites produces beta subunits that remain stably expressed in the endoplasmic reticulum, in association with binding protein but not as irreversible aggregates. The assembly efficiency of nonglycosylated beta subunits is decreased but a reduced number of functional Na+,K+ pumps is expressed at the cell surface. The lack of sugars does not influence the apparent K+ or ouabain affinity of the Na+,K+ pumps. Thus, these data show that disulfide bond formation and N-glycosylation may play important but qualitatively distinct roles in the initial folding of oligomeric protein subunits. Moreover, the results suggest that an endoplasmic reticulum degradation pathway exists, which is glycosylation-dependent.