The erythrocyte membrane was investigated in weanling male rats pair fed with magnesium-deficient and control diets for 8 days. Fluorescence polarization studies revealed a 15% increase in the fluidity of membranes from deficient rats. A similar increase in the fluidity of liposomes indicated that protein was not involved. The change was associated with decreased osmotic fragility of intact erythrocytes; the cells lost their biconcavity and had a flattened appearance with surface irregularities. Analysis of the membranes showed decreased amounts of magnesium, cholesterol, and sphingomyelin in the deficient group. The reduced ratios of cholesterol to phospholipid and sphingomyelin to phosphatidylcholine were consistent with the increased fluidity. Addition of physiological amounts of magnesium to the medium rigidified membranes incubated in tris(hydroxymethyl)-aminomethane buffer, and this was prevented by the presence of EDTA. Cross-incubation experiments with erythrocyte ghosts and plasma from the two groups of rats showed that magnesium-deficient plasma increased the fluidity of control ghosts and control plasma rigidified ghosts from magnesium-deficient rats. Addition of sufficient magnesium chloride to raise the magnesium content of deficient plasma to normal had no significant effect. These results show that the increased fluidity of the erythrocyte membrane in magnesium deficiency is due to physicochemical exchange with the plasma. Although magnesium can directly influence membrane fluidity, the change during its deficiency in vivo is mainly mediated indirectly via disturbances in lipid metabolism.