1. A method was developed for measuring the cytoplasmic magnesium buffering of intact red cells using the divalent cation selective ionophore A23187. Addition of A23187 to a suspension of red cells induces rapid equilibration of ionized magnesium across the cell membrane. 2. Entry of magnesium into red cells is associated with cell swelling and depolarization of the membrane potential. 3. At an external ionized magnesium concentration of about 0.15 mM corresponding to an internal ionized concentration of 0.4 mM the addition of A23187 did not produce a change in the magnesium content of the cells. This indicates that the normal ionized magnesium concentration inside the oxygenated red cell is about 0.4 mM. 4. The magnesium buffering curve for oxygenated, inosine-fed human red blood cells is adequately described by the existence of three buffer systems of increasing capacity and decreasing affinity. These are 0.15 mM with a Km < 10(-7) M, probably structural magnesium bound within the cell proteins; 1.6 mM with a Km approximately equal to 0.08 mM, mainly ATP and other nucleotides; and about 21-25 mM with a Km approximately equal to 3.6 mM, a major portion of this being organic phosphates. It is suggested that the contribution of 2,3-DPG to the low affinity site involves each phosphate group acting as an independent binding site for magnesium.