Band 3 is the most abundant integral protein of the red blood cell membrane^1,2. It performs two critical biological functions: maintaining ionic homeostasis, by transporting Cl^– and HCO₃^– ions, and providing mechanical stability to the erythroid membrane^1,2. Erythroid band 3 (AE1) is one of three anion exchangers that are encoded by separate genes³. The AE1 gene is transcribed by two promoters: the upstream promoter produces erythroid band 3, whereas the downstream promoter initiates transcription of the band 3 isoform in kidney⁴. To assess the biological consequences of band 3 deficiency, we have selectively inactivated erythroid but not kidney band 3 by gene targeting in mice. Although no death in utero occurred, the majority of homozygous mice die within two weeks after birth. The erythroid band 3 null mice show retarded growth, spherocytic red blood cell morphology and severe haemolytic anaemia. Remarkably, the band 3^−/− red blood cells assembled normal membrane skeleton thus challenging the notion that the presence of band 3 is required for the stable biogenesis of membrane skeleton. The availability of band 3^−/− mice offers a unique opportunity to investigate the role of erythroid band 3 in the regulation of membrane-skeletal interactions, anion transport and the invasion and growth of malaria parasite into red blood cells.