Southeast Asian ovalocytosis (SAO) is a hereditary form of elliptocytosis resulting in rigid, oval-shaped erythrocytes resistant to invasion by malaria parasites. The molecular defect is due to deletion of codons 400-408, encoding a 9-amino-acid sequence located at the boundary between the cytosol and the first transmembrane segment in Band 3, the erythrocyte anion transport protein. We have carried out an extensive characterization of Band 3 isolated from SAO erythrocytes which contain about 50% mutant Band 3. A slightly higher proportion of Band 3 in SAO erythrocytes was left associated with the cytoskeleton after extraction of ghost membranes with non-ionic detergents. Size exclusion high performance liquid chromatography analysis showed that SAO Band 3 contained a higher proportion of tetramers relative to dimers (50% tetramer) than normal Band 3 (33% tetramer). The circular dichroism spectrum of Band 3 from SAO erythrocytes was very similar to the spectrum for normal Band 3. Enzymatic deglycosylation and tomato lectin binding showed that SAO Band 3 lacked the polylactosaminyl oligosaccharide found on normal Band 3. SAO Band 3 was unable to bind the anion transport inhibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate, suggesting a dramatic alteration in the inhibitor binding site. In conclusion, deletion of 9 amino acids from Band 3 on the cytosolic side of the membrane affects the properties (glycosylation and inhibitor binding) of Band 3 on the opposite side of the membrane without dramatic changes in the secondary and quaternary structure of the protein.