A complex family of 4.1R isoforms has been identified in non-erythroid tissues. In this study we characterized the exonic composition of brain 4.1R-10-kDa or spectrin/actin binding (SAB) domain and identified the minimal sequences required to stimulate fodrin/F-actin association. Adult rat brain expresses predominantly 4.1R mRNAs that carry an extended SAB, consisting of the alternative exons 14/15/16 and part of the constitutive exon 17. Exon 16 along with sequences carried by exon 17 is necessary and sufficient to induce formation of fodrin-actin-4.1R ternary complexes. The ability of the respective SAB domains of 4.1 homologs to sediment fodrin/actin was also investigated. 4.1G-SAB stimulates association of fodrin/actin, although with an approximately 2-fold reduced efficiency compared with 4.1R-10-kDa, whereas 4.1N and 4.1B do not. Sequencing of the corresponding domains revealed that 4.1G-SAB carries a cassette that shares significant homology with 4.1R exon 16, whereas the respective sequence is divergent in 4.1N and absent from brain 4.1B. An approximately 150-kDa 4.1R and an approximately 160-kDa 4.1G isoforms are present in PC12 lysates that occur in vivo in a supramolecular complex with fodrin and F-actin. Moreover, proteins 4.1R and 4.1G are distributed underneath the plasma membrane in PC12 cells. Collectively, these observations suggest that brain 4.1R and 4.1G may modulate the membrane mechanical properties of neuronal cells by promoting fodrin/actin association.