Human erythroid alpha-spectrin alleles responsible for hereditary elliptocytosis (alphaHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism alphaLELY (LELY: Low Expression LYon) occurs in trans. The alphaLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA --> GTA, Leu --> Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the alpha-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of alpha-chains from the alphaLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif alpha subunit peptides containing the dimer initiation region. The two forms of alpha spectrin produced by alternative mRNA splicing of the alphaLELY allele were represented by alpha18-21(1857), a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and alpha18-21(1857-Delta46), a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with alpha18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the alphaIV and alphaV domains that was the characteristic feature initially used to identify the alphaLELY (SpalphaV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the alpha21 motif, because this region of the alpha18-21(1857-Delta46) peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an alphaLELY homozygote and the two alphaLELY recombinant peptides strongly suggests that little, if any, of the 50% of the alpha chains from the alphaLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant alphaLELY peptides, the inability of detectable amounts of exon 46(-) alpha chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.