Proper folding of the Na,K-ATPase β subunits followed by assembly with the α subunits is necessary for their export from the endoplasmic reticulum (ER). Here we examine roles of the ER lectin chaperone, calnexin, and non-lectin chaperone, BiP, in folding and quality control of the β(1) and β(2) subunits in Madin-Darby canine kidney cells. Short term prevention of glycan-calnexin interactions by castanospermine slightly increases ER retention of β(1), suggesting minor involvement of calnexin in subunit folding. However, both prolonged incubation with castanospermine and removal of N-glycosylation sites do not affect the α(1)-assembly or trafficking of β(1) but increase the amount of the β(1)-bound BiP, showing that BiP can compensate for calnexin in assisting β(1) folding. In contrast to β(1), prevention of either N-glycosylation or glycan-calnexin interactions abolishes the α(1)-assembly and export of β(2) from the ER despite increased β(2)-BiP binding. Mutations in the α(1)-interacting regions of β(1) and β(2) subunits impair α(1) assembly but do not affect folding of the β subunits tested by their sensitivity to trypsin. At the same time, these mutations increase the amount of β-bound BiP but not of β-bound calnexin and increase ER retention of both β-isoforms. BiP, therefore, prevents the ER export of folded but α(1)-unassembled β subunits. These α(1)-unassembled β subunits are degraded faster than α(1)-bound β subunits, preventing ER overload. In conclusion, folding of the β(1) and β(2) subunits is assisted predominantly by BiP and calnexin, respectively. Folded β(1) and β(2) either assemble with α(1) or bind BiP. The α(1)-bound β subunits traffic to the Golgi, whereas BiP-bound β subunits are retained and degraded in the ER.