Anion exchanger 1 (AE1, or Band 3) is an integral membrane glycoprotein found in erythrocytes, responsible for the electroneutral exchange of chloride and bicarbonate ions across the plasma membrane. Southeast Asian ovalocytosis (SAO) results from a nine-amino acid deletion in the first transmembrane segment (TM) of the AE1 protein that abolishes its transport function. The effects of the SAO deletion on: (1) the efficiency of integration of TM1 into the membrane, and (2) the precise positioning of TM1 relative to the membrane were investigated using scanning N-glycosylation mutagenesis in a cell-free transcription/translation system and in transfected HEK293 cells. AE1 or SAO constructs containing either the endogenous N-glycosylation site at Asn642 in extracellular loop 4 (EC4) or single N-glycosylation sites engineered into an expanded extracellular loop 1 (EC1) were used. N-glycosylation efficiency of EC1 in the SAO construct was significantly lower than that of the AE1 construct, indicating that the SAO deletion impairs membrane integration of TM1 and the translocation of EC1 across the membrane. Scanning N-glycosylation mapping of EC1 in the cell-free system and in transfected cells showed that the C-terminus of both AE1 and SAO TM1 were at the same position relative to the membrane. Thus, the SAO deletion is likely to cause a pulling-in of the polar amino acid sequence immediately N-terminal to the deletion into the lipid bilayer, allowing SAO TM1 that was inserted to assume a transmembrane disposition.