To study the changes in the synthesis of the major membrane skeletal proteins, their assembly on the membrane, and their turnover during terminal red blood cell maturation in vivo, we have compared early proerythroblasts and late erythroblasts obtained from the spleens of mice at different times after infection with the anemia-inducing strain of Friend virus (FVA). Metabolic labeling of these cells indicates striking differences between early and late erythroblasts. In early erythroblasts, spectrin and ankyrin are synthesized in large amounts in the cytosol with proportionately high levels of spectrin and ankyrin messenger RNA (mRNA). In contrast, only small amounts of these polypeptides are incorporated into the skeleton, which is markedly unstable. In late erythroblasts, however, the synthesis of spectrin and ankyrin and their mRNA levels are substantially reduced, yet the net amounts of these polypeptides assembled in the membrane skeleton are markedly increased, and the membrane skeleton becomes stable with no detectable protein turnover. The mRNA levels and the synthesis of the band 3 and 4.1 proteins are increased considerably in terminally differentiated normoblasts with a concomitant increase in the net amount and the half-life of the newly assembled spectrin and ankyrin. Thus, the increased accumulation of spectrin and ankyrin at the late erythroblast stage is a consequence of an increased recruitment of these proteins on the membrane and an increase in their stability rather than a transcriptional upregulation. This is in contrast to band 3 and 4.1 proteins, which accumulate in direct proportion to their mRNA levels and rates of synthesis. These results suggest a key role for the band 3 and 4.1 proteins in conferring a long-term stability to the membrane skeleton during terminal red blood cell differentiation.