The human erythrocyte anion-exchange protein (band 3 or AE1) was cloned from a fetal liver cDNA library. Three overlapping clones, encompassing 3637 nucleotides, were analyzed in detail. These encode a 911-amino acid protein (Mr 101,791) and detect a single 4.7-kilobase species in human reticulocyte RNA. The corresponding gene is located on chromosome 17. The protein is similar in structure to other anion exchangers and is divided into three regions: a hydrophilic, cytoplasmic domain that interacts with a variety of membrane and cytoplasmic proteins (residues 1-403); a hydrophobic, transmembrane domain that forms the anion antiporter (residues 404-882); and an acidic, C-terminal domain of unknown function (residues 883-911). The N-terminal domain contains several conserved sections (e.g., residues 57-86, 102-164, 219-347, and 375-403), some of which may contribute to binding sites for ankyrin, protein 4.1, or protein 4.2. The membrane domain is highly conserved with the exception of a single segment (residues 543-567) that contains several sites for cleavage of the protein by extracellular proteases. Based on hydropathy analyses and the wealth of available topographical and functional data, a model is proposed in which the protein crosses the membrane 14 times.