Six individuals with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) from three unrelated families were evaluated. Defects in the ability of spectrin (Sp) to undergo self-association were present, and associated with increased recovery of the Sp alpha I 74-kD fragment after limited tryptic digestion (Sp alpha I/74 variant). Because mutations associated with the Sp alpha I/74 variant described to date have been localized to the 5′ coding region of the alpha-Sp gene (exon 2) or at the 3′ coding end of the beta-Sp gene (exon 30), the polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) method was used to detect mutations in these two regions. In one family with HE, an abnormal pattern of migration of PCR- amplified fragments containing exon 2 was observed, and led to the detection of a new mutation (Ile24Ser) in helix 3 of repeating segment alpha 1. In the two other families, an abnormal pattern of migration of PCR-amplified fragments containing exon 30 was observed in affected individuals, and sequencing led to the identification of two new mutations (Ala2023Val and Trp2024Arg) in helix 1 of repeating segment beta 17. The elliptogenic potential of these mutations emphasizes the importance of the conformational integrity of each of the three helices involved in the formation of the Sp heterodimer contact site, and will help identify critical amino acids involved in this interaction.