The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments crosslinked by α2β2-spectrin tetramers with the assistance of protein 4.1R. Actin and 4.1R bind to the actin-binding domain (βABD) at the N-terminus of the spectrin β-chain. The adjacent, C-terminal end of α-spectrin, contains a calmodulin-like domain (αCML, aa 2262–2418) that is also called the EF hand domain and is thought to be inert or vestigial. However, the sph1J/sph1J mouse, which has severe hereditary spherocytosis and unstable RBC membranes, makes a mutant α-spectrin that lacks the last 13 amino acids (αCMLΔC13), showing that the domain has some important function. To investigate this function we “fished” for interacting proteins using glutathione-S-transferase (GST)-fused to the CML domain—either the wildtype (αGST-CML) or sph1J (αGST-CMLΔC13). αGST-CML retrieved protein 4.2 from a 2M Tris HCl extract of spectrin-actin depleted human RBC membranes. Protein 4.2 bound αGST-CML with high affinity (Kd = 2.7 x 10−7M) but did not bind αGST-CMLΔC13. Binding was abolished by 1 mM Ca2+, which converts the CML domain to the liganded conformation. The binding site on protein 4.2 localized, at least partly, to amino acids 411–492. Because red cells lacking protein 4.2 are not as severely affected as sph1J/sph1J RBCs, we also tested the effect of the αCMLΔC13 mutation on spectrin-actin binding. A minispectrin was prepared containing the actin-binding domain plus the first four spectrin repeats of the β-chain, combined with the CML domain (±ΔC13) and the last four repeats of the α-chain. The normal and mutant minispectrins were incubated with protein 4.1R, F-actin, or both proteins. The results were striking. The minispectrin containing the normal CML domain bound actin in the presence of protein 4.1R, but the minispectrin containing the mutant CML domain did not. Similarly, the mutant minispectrin was defective in its ability to bind 125I-4.1R in the presence of a constant amount of F-actin. However, the mutation did not affect binding of the minispectrin to protein 4.1R in the absence of actin. We have not yet tested whether protein 4.2 or Ca2+ modulate the effects of the CML domain on spectrin-actin binding. In summary, these experiments clearly show that the calmodulin-like (EF hand) domain of α-spectrin, which was previously considered inert, binds protein 4.2 and also contributes to spectrin-actin binding in the presence of protein 4.1R. Further experiments will be needed to determine whether the CML domain binds actin directly or strengthens the binding of the adjacent actin-binding domain.