Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R135) and an 80 kDa isoform (4.1R80), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R135 isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R80 isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R135 isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R80. In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R135 and 4.1R80 bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R80 was Ca2+-independent, whereas the binding of 4.1R135 was strongly Ca2+-dependent. The HP of 4.1R135 mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R135 to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R135 isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.