Band 3 is a typical polytopic membrane protein that mediates anion exchange activity [anion exchanger 1 (AE1)]. Although the topology and topogenesis of approximately 40 residues just after transmembrane (TM) 9 have been extensively studied, the topogenesis of this region [tenth region (10thR)] has been unclear. Glycosylation sites created in the 10thR were efficiently glycosylated in a cell-free transcription/translation system, whereas the glycosylation efficiencies were quite low in a cultured cell system. When TM12-14 was deleted or when cycloheximide was added to the culture medium, however, the glycosylation efficiency in the cultured cells increased to the same level as in the cell-free system, indicating that TM12 is essential for the sequestration from oligosaccharyl transferase into membrane and that cycloheximide treatment of the cells can mimic the cell-free system by reducing the rate of chain elongation. The glycosylation efficiency in cultured cells also increased with deletion of TM1-3. These results suggest that the 10thR is transiently extruded into the lumen and then inserted into the membrane. Both TM12 and the distant TM1-3 affect the membrane insertion of the 10thR. This indicates that during the folding of the protein, the 10thR is inserted into the membrane after the TM1-12 segments are properly assembled.