Brain spectrin tetramer was purified from pig brain membranes in milligram quantities. The tetramer had subunits of Mr = 265,000 (alpha) and Mr = 260,000 (beta), Rs = 21.4 nM, S20,w = 11 S, V = 0.725 ml/g, frictional ratio of 2.9, and calculated molecular weight of 9.7 x 10(5). The subunits were isolated in greater than 95% purity by chromatography on hydroxylapatite in 7 M urea, as described for erythrocyte spectrin (Calvert, R., Bennett, P., Gratzer, W. (1980) Eur. J. Biochem. 107, 355-361). Peptide maps of the subunits revealed few if any common peptides. The subunits were visualized by rotary shadowing as single-stranded flexible rods 100 nm in length with no homodimers by lateral or end association. When the subunits were renatured together, double-stranded tetramers 200 nm in length were formed, as well as higher oligomers. These results indicate that spectrin tetramers are formed by laterally associated alpha,beta dimers attached by head-to-head linkage of each alpha chain with a beta chain. The reassembled subunits regained the ability to increase the low shear viscosity of actin, although isolated alpha or beta subunits were inactive. Hybrid molecules were formed with brain alpha and pig erythrocyte spectrin beta subunit and were visualized as double-stranded rods 100 nm long with no tetramers. 125I-labeled brain alpha chain that was hybridized with erythrocyte beta subunit acquired the ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain alpha chain bound only to beta subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate gels. Affinity-purified antibodies against brain spectrin cross-reacted with both subunits of erythrocyte spectrin and with polypeptides of a similar molecular weight to brain spectrin in membranes of all tissues examined. Cross-reacting polypeptides were localized exclusively in plasma membranes of subcellular fractions from liver. These studies provide strong additional evidence that brain and mammalian erythrocyte spectrin belong to a closely related family of proteins with conserved functions in both subunits.