An improved method for the large scale isolation of Band 3 has been developed and used to isolate peptides from the cytoplasmic portion of the molecule. Membranes were treated with 33 mM lithium 3,5-diiodosalicylate to remove “extrinsic” membrane proteins and then solubilized in 2% sodium dodecyl sulfate. An activated thiol column was used to separate the sialoglycoproteins from Band 3 and the latter was purified by gel filtration on Sepharose 6B in sodium dodecyl sulfate. A chymotryptic fragment of Band 3 (M, 58,000; chymotryptic Band 3), produced by digestion of intact cells, and a tryptic fragment of Band 3 WI, 49,000; tryptic Band 3), produced by digestion of membranes were purified similarly. Each purified component was cleaved by S-cyanylation (Jacobson, G. R., Schaffer, M. H., Stark, G. R., and Vanaman, T. C. (1973) J. Biol. Chem. 248, 6583-6591) in 7.5 M guanidine hydrochloride. Intact and chymotryptic Band 3 produced three peptides (iM, 36,000, 24,000, and 12,000) which appeared to be homogeneous when analyzed by gel electrophoresis in sodium dodecyl sulfate or acid-urea. Lactoperoxidase-catalyzed iodination indicated that these peptides are derived from the cytoplasmic side of membrane. These peptides were purified by CM-cellulose and Sephadex G-100 and found to be rich in glutamate (or glutamine) and did not contain detectable sugars. Antibodies prepared against Band 3 were found to be directed against the cytoplasmic segment of Band 3 and these were used to show that the 12,000and 24,000-dalton peptides were derived from the 36,000-dalton piece. Cleavage of intact Band 3 by a two-step cyanylation reaction using K’%N indicated that the 36,000dalton peptide and the 24,000-dalton peptide were both derived from the NH,-terminal end. These experiments support the idea that a 40,000-dalton segment of Band 3 bearing the NH, terminus is on the cytoplasmic side of the lipid bilayer. These results are also consistent with the view that the bulk of the Band 3 material is a homogeneous polypeptide chain.