Phosphorus-31 nuclear magnetic resonance (NMR) spectra were determined from intact erythrocytes of a patient with homozygous beta-thalassemia and from three patients with pyruvate kinase (PK) deficiency. The intracellular 2,3-diphosphoglycerate-(2,3-DPG) were mildly elevated in the thalassemia patient and in PK heterozygotes, and were markedly elevated in the PK-deficient homozygotes. The 2,3-DPG chemical shifts in the patients' NMR spectra were consistently positioned at a higher magnetic field than normal. When the patients with thalassemia or PK deficiency were transfused with blood from a normal donor, or when a patient's erythrocytes were mixed with normal erythrocytes, two distinct sets of 2,3-DPG resonances appeared in the resulting spectra, allowing the simultaneous quantification of the 2,3-DPG levels in each erythrocyte population. The ability of NMR to detect heterogeneous cell populations may be useful for the diagnosis of congenital hemolytic anemias in patients who have been transfused.