The demonstration that red cell levels of 2,3-DPG play a central role in determining the affinity of hemoglobin for oxygen has resulted in a renewed interest in methods for maintaining or restoring the level of this organic phosphate in stored blood. The effects of addition of inorganic phosphate, inosine and pyruvate, individually or in various combinations, all in a final concentration of 10 mM were evaluated 1 and 4 hours after supplementation of ACD-stored blood, 21 to 28 days old. In 14 studies the initial 2,3-DPG level averaged 176 mµmoles/ ml. RBC. In normal fresh blood the 2,3-DPG was 4200 ± 400 mµmoles/ml. RBC. Inosine addition raised the 2,3-DPG to 1395, inosine and phosphate to 1528, inosine and pyruvate to 3363, while the combination of inosine, pyruvate and phosphate increased the level to 6637 mµmoles/ml. RBC. After 2-3 hours of incubation most of the 2,3-DPG restoration had occured. The maximum effects did not require prior pH correction of the blood. Although inosine in a final concentration of 10 mM was required for optimum effects, the phosphate and pyruvate concentrations could be reduced to 4 mM. In the presence of inosine and phosphate alone the red cell accumulated large quantities of triose phosphates, fructose diphosphate and glucose-6-phosphate. These levels were reduced by the addition of pyruvate. Pyruvate addition appears necessary to provide sufficient NAD for maximum 2,3-DPG synthesis. Associated with 2,3-DPG restoration of the stored blood there was a rise in the P50 (the oxygen tension at which hemoglobin is 50% saturated) from a mean of 16.7 to 31.6 mm. Hg.