Erythrocyte membrane structural parameters were studied in transfusion-dependent beta-thalassemia patients, in long-term transfused patients (regularly transfused < 15 years), and in those who had not yet obtained transfusions. Controls were voluntary students up to 30 years of age without diagnosis or clinical signs of thalassemia. Membranes were isolated and investigated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron paramagnetic resonance (EPR) spectroscopy. Data obtained from the thiol-reactive spin label N-ethyl-maleimidoproxyl reveal immobilization of protein environment in erythrocyte membranes from thalassemic patients. SDS-PAGE shows both degradation and aggregation of membrane proteins. Thalassemic erythrocyte membranes exert higher order parameters in the hydrophobic region as determined by 16-doxyl-stearic acid. Rotational correlation times of this spin label increase only in transfused patients. Polarity is higher in membranes of all patients than in controls. In the polar interface, order parameters obtained from 5-doxyl-stearic acid increase in non-transfused and decrease in transfusion-dependent patients as compared with controls. Transfused patients exert increasing membrane order in the hydrophobic region and counter-currently decreasing order in the polar interface indicating loss of membrane integrity along with the loss of fluidity and polarity gradients and the loss the energetic barrier function of the membrane.