Erythrocyte ankyrin contains an 89-kDa domain (residues 2-827) comprised almost entirely of 22 tandem repeats of 33 amino acids which are responsible for the high affinity interaction of ankyrin with the anion exchanger (Davis, L., and Bennett, V. (1990) J. Biol. Chem. 265, 10589-10596). The question of whether the repeats are equivalent with respect to binding to the anion exchanger was addressed using defined regions of erythrocyte and brain ankyrins expressed in bacteria. The conclusion is that the repeats are not interchangeable and that the 44 residues from 722 to 765 are essential for high affinity binding between erythrocyte ankyrin and the anion exchanger. Residues 348-765 were active whereas a polypeptide of the same size (residues 305-721) but missing the 44 residues was not active. The difference between the active and inactive polypeptides was not caused by the degree of folding based on circular dichroism spectra. The 44 residues from 722 to 765 were not sufficient for binding since deletions of residues from 348 to 568 resulted in a 10-fold loss of activity. However, the role of residues 348-568 may be at the level of folding rather than a direct contact since the deleted sequences were not active in the absence of 722-765 and since circular dichroism spectra revealed significant loss of structure in the smaller polypeptides. Further evidence that the 33-residue repeats are not equivalent in ability to bind to the anion exchanger is that a region of human brain ankyrin containing 18 33-residue repeats with 67% overall sequence identity to erythrocyte ankyrin was 8-fold less active than a region of erythrocyte ankyrin containing only 12 repeats. The fact that the anion exchanger binds to certain repeats suggests that the other 33-amino acid repeats could interact with proteins distinct from the anion exchanger and provide ankyrin with the potential for considerable diversity in association with membrane proteins as well as cytoplasmic proteins. Tubulin was identified as one example of a protein that can interact with ankyrin repeats that are not recognized by the anion exchanger.