Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the previously identified spectrin-actin binding domain (10 kDa molecular mass) of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, denoted 10k21 and 10k19,21, were able to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette, 10k19 and 10k0, did not function in these assays. A bacterially expressed fusion protein with glutathione-S-transferase, designated GST-10k21, congealed spectrin-actin into a network in vitro as found with purified protein 4.1. Additionally, incorporation of GST-10k21 into mechanically weak, 4.1-deficient membranes increased mechanical strength of these membranes to normal. GST-10k19 did not function in these assays. These results show that the 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 are suggested to have different, yet to be defined functions.