Ankyrin is an important key protein transferring the signal between the inside and outside of eukaryotic cells, because of its ability to bind both to ionic channels of the plasma membranes and to cytoskeletal proteins. In this study, we investigated new ankyrin binding proteins in rat cerebral membrane. Five main proteins in the extract of the demyelinated membranes were bound to erythrocyte ankyrin as examined by affinity chromatography. One of the proteins had a molecular weight of 97 kD that was almost identical with that of the alpha-subunit of Na+, K(+)-ATPase. 125I-labeled erythrocyte ankyrin was bound to the alpha-subunit of cerebral Na+, K(+)-ATPase, which contains both alpha and alpha(+) subunits. The binding experiment also showed that 70% of the total erythrocyte ankyrin bound to cerebral Na+, K(+)-ATPase. On the other hand, erythrocyte ankyrin binds less to Na+, K(+)-ATPase prepared from rat brain stem axolemma which contained only alpha(+) subunit. These results suggest that erythrocyte ankyrin may bind with high affinity to a cerebral Na+, K(+)-ATPase isoform with alpha subunit, but with much lower affinity to axonal Na+, K(+)-ATPase containing alpha(+) subunit.