Efficacy in the diagnosis of hereditary spherocytosis (HS) using a system of dual-angle differential light scattering of a monochromatic laser beam on sphere-shaped and fixed erythrocytes was evaluated. Fifty-one nonsplenectomized patients with HS, 6 with autoimmune hemolytic anemia, and 140 control subjects were studied. The percentage of erythrocytes with hemoglobin concentration more than 410 g/L (hyperhemoglobin) was significantly different in patients with HS and control subjects (P < 0.001). Hyperhemoglobin could be detected even in the mildest HS, in all patients with autoimmune hemolytic anemia, and in some control subjects, although none of the latter had more than 3%. This technique is extremely sensitive to the presence of spherocytes and the absence of erythrocytes with more than 410 g/L hemoglobin excludes HS. The presence of hyperhemoglobin is not specific for HS and increased amounts of hyperhemoglobin were found in patients with autoimmune hemolytic anemia and control subjects. To make a confident diagnosis of HS, the detection of hyperhemoglobin must be evaluated in the same clinical setting and with the same criteria as those applied to the osmotic fragility test.