We have previously reported that human erythrocyte band 3 contains 90-95% of the reconstitutable glucose transport activity of the erythrocyte membrane (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33). We have now found that monoclonal and polyclonal antibodies to epitopes on band 3 specifically removed band 3 and more than 90% of the reconstitutable glucose transport activity from unfractionated octylglucoside extracts of erythrocyte membranes; nonimmune serum removed neither. Western blots of whole membrane extracts revealed that the polyclonal antibody to band 4.5 used to isolate cDNA clones presumed to code for the transporter (Mueckler, M., Caruso, C., Baldwin, C.A., Pancio, M., Blench, J., Morris, H.B., Allard, W.J., Lienhard, G.E. and Lodish, H.F. (1985) Science 229, 941-945) reacts strongly with six discrete bands in the 4.5 region. A monoclonal antibody to band 3 also reacts with a Mr 55,000 component of band 4.5. We conclude that band 3 contains the major glucose transporter of human erythrocytes, and that the transport activity in band 4.5 might be attributable to a band 3 fragment. Band 3 is probably a multifunctional transport protein responsible for transport of glucose, anions, and water.