The relationships among Hb concentration, red cell 2,3-diphosphoglycerate (2,3-DPG) and p50 [O2 tension at 50% saturation of Hb] were studied in 20 chronically hypertransfused patients with thalassemia major. In the nontransfused control group, which included normal individuals as well as patients with sickle cell disease or Fe deficiency anemia, the Hb correlated inversely with both 2,3-DPG concentration and p50, as is well established. In contrast, however, prior to transfusion, at the nadir of Hb, patients with thalassemia major had inappropriately low 2,3-DPG concentrations and p50. These findings occurred in all patients, regardless of whether they had received packed, leukocyte-poor, or frozen-thawed red cells. That the time of blood storage was a factor was excluded by repeatedly transfusing 1 patient with packed red cells administered within 4 h of collection of CPDA-1. That red cell function might be impaired by the Fe overloaded thalassemic environment, was excluded by studying a newly diagnosed, newly transfused patient with aplastic anemia. In both cases, the same inability to appropriately increase 2,3-DPG and p50 as the Hb fell during the intertransfusion interval was noticed. Red cells of chronically transfused patients apparently are unable to adapt to the decline in Hb that occurs during the intertransfusion interval.