Quality control of protein biosynthesis requires ER-retention and ER-associated degradation (ERAD) of unassembled/misfolded molecules. Although some evidence exists for the organization of the ER into functionally distinct membrane domains, it is unknown if such domains are involved in the retention and ERAD of unassembled proteins. Here, it is shown that unassembled MHC class I molecules are retained in the ER without accumulating at ER-exit sites or in the ERGIC of β2m−/−cells. Furthermore, these molecules did not cluster in the ER membrane and appeared to be highly mobile even when ERAD or their association with calnexin were inhibited. However, upon ATP depletion, they were reversibly segregated into an ER membrane domain, distinct from ER exit sites, which included calnexin and COPII, but not the ERGIC marker protein p58. This quality control domain was also observed upon prolonged inhibition of proteasomes. Microtubules were required for its appearance. Segregation of unfolded proteins, ER-resident chaperones, and COPII may be a temporal adaptation to cell stress.