Further similarity between mammalian erythrocyte spectrin and pig brain spectrin has been demonstrated by (a) formation of hybrid molecules with brain alpha-chains and erythrocyte beta-chains and by (b) identification of an ankyrin protein in brain membranes. Hybrid spectrin molecules prepared from brain alpha-chains and erythrocyte beta-chains were visualized by low-angle rotary shadowing as double-stranded rods (dimers) 100 nM in length. 125I-labeled brain alpha-chain that was hybridized with erythrocyte beta-subunit acquired 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 (SDS) gels. Thus brain spectrin and mammalian erythrocyte spectrin have shared functional sites involved in association of their subunits. Additional evidence for similarity of brain and erythrocyte membranes is the finding of a 210,000 Mr membrane protein in brain that cross-reacts with erythrocyte ankyrin and has a water-soluble domain of 72,000 Mr that is produced by protease digestion. The 72,000 Mr domain of brain ankyrin has been isolated by affinity chromatography on erythrocyte spectrin-Sepharose, and was demonstrated to bind directly to erythrocyte and brain spectrin. The brain 72,000 Mr fragment has distinct peptide maps from the erythrocyte 72,000 Mr ankyrin fragment and thus is not a result of erythrocyte contamination.