Expression of the complex gene encoding multiple isoforms of structural protein 4.1 is regulated by alternative pre-mRNA splicing. During erythropoiesis, developmental stage-specific inclusion of exon 16 generates protein 4.1 isoforms having a fully functional spectrin-actin binding domain. Here we show that human mammary epithelial cells (HMEC), coincident with the dramatic morphological changes induced by altered culture conditions, exhibit a novel pre-mRNA splicing switch involving a new exon (exon 17B, 450 nucleotides) in the COOH-terminal coding region. 4.1 RNA expressed in proliferating HMEC adherent to culture dishes mostly excluded exon 17B, whereas 4.1 transcripts processed in nondividing suspension cultures of HMEC strongly included this exon. This pre-mRNA splicing switch was reversible: cells transferred from poly(2-hydroxyethyl methacrylate) back to plastic resumed cell division and down-regulated exon 17B expression. More detailed studies revealed complex tissue-specific alternative splicing of exon 17B and another new exon 17A (51 nucleotides). These results predict the existence of multiple 4.1 protein isoforms with diverse COOH termini. Moreover, they strongly suggest that regulation of gene expression during differentiation of epithelial cells is mediated not only by transcriptional mechanisms, but also by post-transcriptional processes such as alternative pre-mRNA splicing.