Using immunoelectron microscopy and isoform-specific antibodies against Na,K-ATPase to study changes in Na,K-ATPase in rat erythroblastic cells during maturation, we unexpectedly observed numerous antigenic sites against the alpha3-isoform in the cytoplasmic phase. There was an increase in the number of alpha3-isoforms after denucleation of the erythroblast. The increase was transient. As the reticulocyte matured into a red blood cell, the number of alpha3-isoforms was reduced drastically. This alpha3-isoform was distributed in a reticular pattern resembling the double layers of endoplasmic reticulum. Western blot analysis confirms the presence of the alpha3-isoform in these cells. X-ray microanalysis of the erythroid series of cells in the bone marrow shows that sodium concentration in the young reticulocyte is higher than that in the nucleated erythroblast. The reason for the transient increase in this pump protein is not clear. It is possible that the increase in sodium concentration in the reticulocyte plays a role in the increase in pump protein synthesis.