We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M k M k cells) or GPA (En(a؊) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to ϳ60% of that in normal control red cells as a result of an increased apparent K m for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V max for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K d for iodide binding, in agreement with the unchanged K m found in transport studies. However, the apparent diffusion quenching constant (K q ) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V max observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.The anion exchange protein AE1 (band 3) and glycophorin A (GPA) 1 are both present in the human red cell membrane at ϳ1 ϫ 10 6 copies/cell. Evidence suggests that these two proteins associate both in internal cell membranes during their biosynthesis and in red cell membranes (1-12). Reduction of the amount of band 3 in the human red cell membrane results in membrane defects, and the almost total absence of band 3 in man compromises life and requires intervention (13,14). However, GPA can be reduced or even absent as a result of genetic defects without known clinical consequences (15). In some individuals, GPA is completely absent in the red cell membrane (En(aϪ) cells), whereas other individuals have red cells that lack both GPA and glycophorin B (GPB) (M k M k cells). In other cases, only GPB is absent in the red cell membrane (S Ϫ s Ϫ U Ϫ cells). The GPA and GPB genes are located in tandem on chromosome 4, and these two highly homologous genes are prone to deletions, gene conversions, and crossovers.The structure and function of band 3 are altered in En(aϪ) red cells (16,17) and M k M k red cells (18 -20). In these cells, band 3 has a higher apparent molecular weight due to an increase in the size of the N-glycan chain (16,17,20). In M k M k cells, the sulfate transport activity of band 3 is ϳ60% of that in normal red cells as a result of an increase in the apparent K m for sulfate (20). It has been argued that this altered transport activity is due to the lack of GPA in M k M k cells, as there is no evidence for an interaction between band 3 and GPB.The transport domain of band 3 has been studied by the fluorescence quenching of eosin-5-maleimide (EMA)-labeled...