Hereditary spherocytosis (HS) is attributed to red blood cell membrane protein defects, caused by mutations in ankyrin, spectrin, band 3 and protein 4.2. In this study, the presence of band 3 mutations was investigated in a patient presenting mild HS and band 3 deficiency. Using single strand conformation polymorphism analysis, a shift in exon 16 of the band 3 gene was found. DNA sequencing revealed a point mutation 2102 T > C, changing methionine at position 663 to lysine. The M663K substitution was not found in either the parents or in the siblings, and the restriction fragment length polymorphism analysis of 100 alleles from a random Brazilian population did not reveal this mutation, suggesting that this gene defect is more likely to be a de novo mutation, causing HS. Flow cytometry of eosin-5-isothiocyanate (EITC)-labelled erythrocytes showed, in the patient, 54% of band 3 protein content vs. 78% based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, suggesting that flow cytometry is a more sensitive method and may be used as a diagnostic tool in membrane disorders related to band 3 deficiency. The characterisation of novel AE1 mutations is helpful to improve the understanding of the role of band 3 protein in cell physiology.745396401