The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.