Red cell aldolase activity decreases normally with cell age at a mathematically predictable rate. Compared to aldolase activity so predicted for red cells of the same age, activity of this glycolytic enzyme was 35 per cent deficient (range 18–53 per cent) in 20 splenectomized patients from eight unrelated families with typical hereditary spherocytosis. In two unsplenectomized patients with hereditary spherocytosis red cell age was estimated from measurement of their diisopropyl‐fluorophosphate (DF³²P)‐labelled red cell survivals, and deficiencies of 62 and 55 per cent in red cell aldolase activity observed. The rates of glycolysis in intact cells and haemolysates measured under conditions designed to produce maximum rates of lactate formation were 20–30 per cent less than expected for the red cell age in the splenectomized subjects. Since patients with hereditary spherocytosis are probably heterozygous for the genetic defect presumed to be present in this disease, the magnitude of deficiency was compared to those reported in heterozygotes for inherited red cell abnormalities such as sickle haemoglobinopathy, glucose‐6‐phosphate dehydrogenase deficiency and pyruvate kinase deficiency and it was found to be similar. This suggests that a deficiency in red cell aldolase activity may be the basic biochemical lesion in hereditary spherocytosis. One of the products of the aldolase reaction, dihydroxyacetone phosphate, is known to be an important precursor for phospholipid in other tissues, suggesting a possible relationship between red cell aldolase deficiency and the known red cell membrane abnormalities of hereditary spherocytosis.