The self-polymerization of alpha beta-spectrin heterodimers to form tetramers and higher oligomers is central to its role as a membrane stabilizer and organizer. Mutations near the amino terminus of alpha I sigma 1-spectrin or the COOH terminus of beta I sigma 1-spectrin often lead to profound impairment heterodimer polymerization and to hemolytic disease of varying severity. Previous studies using an 80-kDa univalent fragment of alpha I sigma 1-spectrin have established that the amino-terminal segment of alpha I sigma 1-spectrin mediates the association of the alpha subunit with either intact heterodimers or with isolated beta-spectrin (beta I sigma 1). However, the nature of the self-association site in beta-spectrin has remained unclear. In the present study, native beta-spectrin and recombinant beta-spectrin peptides representing COOH-terminal portions of two alternative transcripts of the gene on chromosome 2 (beta I sigma 1 or "erythrocyte" spectrin and beta I sigma 2 or "muscle" spectrin), and one transcript of the gene on chromosome 14 (beta II sigma 1 or "beta G-fodrin") have been examined for their ability to bind either intact alpha beta-spectrin or the alpha I-spectrin 80-kDa univalent fragment. Deletion of the nonhomologous beta-spectrin sequence downstream of repeat 17 (spectrin domain III) had no discernible effect on binding. Truncations proximal to codon 2085 of beta I sigma 1-spectrin demonstrated a precipitous loss of activity, accounted for by a loss of both binding affinity and capacity. Further truncations to repeat 16 (codon 1979) restored binding activity to levels approximating that of the intact molecule. Repeat 16/17 and 17/16 chimeras displayed reduced binding activity. Collectively, these data indicate that the beta-subunit self-association site is highly sensitive to conformation, involves widespread interactions within the 17th repeat unit, is largely independent of sequences in domain III, and can be recreated by the deletion of all residues distal to the COOH end (codon 1979) of the 16th and presumably other spectrin sequence repeat units. All beta-spectrins appear to use this binding motif, regardless of the nature of the nonhomologous sequence in domain III.