Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed a greater than 70-kb genomic deletion, beginning within intron 1 and ending within a 1.3-kb region upstream from exon 13. This deletion encompassed both erythroid and nonerythroid translation initiation sites. It accounts for the largest deletion known in genes encoding proteins of the red blood cell membrane. The corresponding mRNA was shortened by 1727 bases, due to the absence of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a similar pattern in lymphoblastoid cells as in reticulocytes. Differential splicing of exons within the undeleted region remained regulated in a tissue-specific manner. Exons 14, 15, and 17a were absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was present in reticulocytes but absent from lymphocytes. Thus, differential splicing on a local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.