This study was designed to make precise the nature and the mechanism of the anemia induced by dietary copper (Cu) deficiency. Male Wistar rats were pair fed from weanling for 6 wk either a Cu-deficient or a control diet. The reduced red blood cell (RBC) 51Cr survival indicates an increased destruction of RBC during Cu deficiency. 1,6-Diphenyl-1,3,5-hexatriene fluorescence polarization studies revealed an increase in the fluidity of erythrocyte membranes from deficient rats. The reduced cholesterol-to-phospholipid ratio was consistent with the increased fluidity. Other results indicate an increased vulnerability of RBC to hemolysis in dilute hydrogen peroxide and an increased formation of lipid peroxidation products. Before exposure to free radical stress, electron spin resonance studies in intact RBC revealed decreased correlation time of 16-doxyl-stearic acid, confirming a more fluid membrane in RBC from Cu-deficient rats. After in vitro peroxidation, RBC from Cu-deficient rats showed a more ordered state of membrane lipids compared with controls. Together, these studies demonstrate the hemolytic nature of the anemia. The shortened survival of erythrocytes apparently results from changes in membrane fluidity and enhanced susceptibility to peroxidation.