Erythroid differentiation is accompanied by dramatic alterations in morphology and membrane mechanical properties resulting, in large part, from reorganization of the membrane skeletal protein network. The 80-kD protein 4.1 is an important organizational component of this membrane skeleton. Recently, it has been recognized that multiple structural isoforms of 4.1 are encoded by a single gene via alternative pre-mRNA splicing, and that an upstream ATG can be spliced in and used for translation of high molecular weight 4.1. We are exploring the hypothesis that differentiation-associated switches in protein 4.1 structure play an important role in membrane reorganization. To study changes in 4.1 gene expression during normal human differentiation, we analyzed 4.1 protein and mRNA structure at various developmental stages. Using immunofluorescence microscopy, we observed high molecular weight 4.1 isoforms in preproerythroblasts producing punctate, predominantly cytoplasmic staining with a perinuclear area of intense fluorescence, while mature red cells expressed very little high molecular weight 4.1. Isoforms containing an alternatively expressed 102-nucleotide exon near the COOH terminus were abundant in both preproerythroblasts and mature cells but produced a punctate distribution of fluorescence over the entire preproerythroblast and intense membrane-associated fluorescence in the erythrocyte. Characterization of RNA by polymerase chain reaction and nuclease protection assays revealed a differentiation-associated switch in pre-mRNA splicing in the spectrin-actin binding domain. Since this domain plays a critical role in regulating membrane material properties, we speculate that this switch may be crucial to reorganization of the skeletal network during erythropoiesis. We conclude that 4.1 isoforms are differentially expressed and differentially localized during erythropoiesis, and that this isoform family is likely to have diverse functions during terminal differentiation.