Spectrin tetramers of the membranes of enucleated mammalian erythrocytes play a critical role in red blood cell survival in circulation. One of the spectrins, aI, emerged in mammals with enucleated red cells after duplication of the ancestral a-spectrin gene common to all animals. The neofunctionalized aI-spectrin has moderate affinity for bI-spectrin, whereas aIIspectrin, expressed in nonerythroid cells, retains ancestral characteristics and has a 10-fold higher affinity for bI-spectrin. It has been hypothesized that this adaptation allows for rapid make and break of tetramers to accommodate membrane deformation. We have tested this hypothesis by generating mice with high-affinity spectrin tetramers formed by exchanging the site of tetramer formation in aI-spectrin (segments R0 and R1) for that of aII-spectrin. Erythrocytes with aIIbI presented normal hematologic parameters yet showed increased thermostability, and their membranes were significantly less deformable; under low shear forces, they displayed tumbling behavior rather than tank treading. The membrane skeleton is more stable with aIIbI and shows significantly less remodeling under deformation than red cell membranes of wild-type mice. These data demonstrate that spectrin tetramers undergo remodeling in intact erythrocytes and that this is required for the normal deformability of the erythrocyte membrane. We conclude that aI-spectrin represents evolutionary optimization of tetramer formation: neither higher-affinity tetramers (as shown here) nor lower affinity (as seen in hemolytic disease) can support the membrane properties required for effective tissue oxygenation in circulation.