An increasing body of experimental evidence demonstrates that intracellular hemoglobin concentration and composition is a primary determinant of pathophysiology in sickle cell disease. To quantitate more precisely the heterogeneous distribution of intracellular hemoglobin concentrations in a given individual with this disease, we have calibrated the phthalate ester separation technique by using discontinuous Stractan density gradients to isolate subpopulations of red cells of relatively uniform corpuscular hemoglobin concentration values. We find that blood from individuals with sickle cell anemia exhibits a markedly broader distribution of corpuscular hemoglobin concentration values, containing both very light and very dense cells, than the red cell density profile from normal individuals. This increased breadth of cell densities in patients with sickle cell anemia remains even after exclusion of the very light and very dense subpopulations. In patients with stable sickle cell anemia, there appears to be minimal variation in the distribution of cell densities that are unimodal but skewed toward higher density values. The phthalate ester method can conveniently be used to follow changes in cell densities during vaso-occlusive events, to monitor therapy targeted at modifying intracellular hemoglobin S concentrations, and in sequential applications in large field trials designed to determine the relationship between red cell heterogeneity and specific manifestations of the sickle cell syndromes.