Band 3 is the major, membrane-spanning, .apprx. 90,000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, this protein was cleaved in situ into large fragments and their disposition ascertained. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38,000 and 55,000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they were anchored in the apolar core of the membrane. Both pieces communicated with the extracellular space, and the 55,000 dalton species extended to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17,000 dalton species, a segment of the 55,000 dalton fragment, which spanned and was firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52,000 daltons and released major band 3 fragments of .ltoreq. 41,000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which was induced only at the cytoplasmic surface, cross-linked intact band 3 and certain of its water-soluble fragments. Finally, fragments of 23,000 daltons were generated from the inner-surface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.