Marked variations in the clinical manifestations of hereditary spherocytosis (HS) have long been recognized. However, neither the molecular nor the cellular basis for this variable expression has been fully delineated. To better define the cellular basis for variable expression of the disease, we evaluated the pathobiology of red cells in a large series of 55 non-splenectomized and 31 splenectomized patients with HS. Red cell membrane surface area, surface area-to-volume ratio, cell volume, and state of cell hydration were quantitated. We found that decreased membrane surface area was a distinguishing feature of red cells in all patients studied, whereas decreased surface area-to-volume ratio as reflected by increased osmotic fragility was noted in only 66% of the non-splenectomized patients. In terms of red cell indexes, the percentage of microcytes was not a good discriminator of HS phenotype but was the best indicator of the severity of the disease. In contrast, the presence of increased numbers of hyperdense cells was an effective discriminating feature of the HS phenotype but a poor indicator of disease severity. These findings have enabled us to define the dominant cellular changes that account for the variable clinical severity of this common red cell membrane disorder and have allowed development of improved approaches for its diagnosis.