AE1 [anion exchanger 1, also known as SLC4A1 (solute carrier family 4, anion exchanger, member 1) and band 3 (erythrocyte membrane protein band 3)] is a major membrane glycoprotein expressed in human erythrocytes where it mediates the exchange of chloride and bicarbonate across the plasma membrane. Glycophorin A (GPA) is a sialoglycoprotein that associates with AE1 in erythrocytes forming the Wrb (Wright b) blood group antigen. These two integral proteins may also form a complex during biosynthesis, with GPA facilitating the cell surface expression of AE1. This study investigates the interaction of GPA with AE1 in K562 cells, a human erythroleukaemic cell line that expresses GPA, and the role of GPA in the cell surface expression of AE1. In K562 cells, GPA was dimeric and N- and O-glycosylated similar to erythroid GPA. GPA was localized at the cell surface, but also localized to the Golgi. AE1 expressed in K562 cells contained both complex and high-mannose oligosaccharides, and co-localized with GPA at the cell surface and in the endoplasmic reticulum (ER). The Wrb antigen was detected at the cell surface of AE1-transfected K562 cells, indicating the existence of an AE1-GPA complex. Immunofluorescence and co-immunoprecipitation studies using AE1 and an ER-localized hereditary spherocytosis mutant (R760Q AE1) showed that GPA and AE1 could interact in the ER. GPA knockdown by shRNAs (small-hairpin RNAs), however, had no effect on the level of cell surface expression of AE1. The results indicate that AE1 and GPA form a complex in the ER of human K562 cells, but that both proteins can also traffic to the cell surface independently of each other.