This is a report of a newly devised method of biochemical modification and freeze-preservation of red blood cells that is considerably less expensive than previous methods and carries a lower risk of contamination. The “rejuvenated” red blood cells are of superior quality, with excellent viability and function after freeze-preservation with 40% w/v glycerol and postthaw washing and storage. At the time of collection, the plastic bag is labeled with the blood type and identification number of the donor, and the label remains affixed during frozen storage. This polyvinyl chloride plastic bag contains integrally attached transfer packs, for blood collection, component separation, red blood cell biochemical modification, freezing, storage, and postthaw dilution before washing. The rejuvenated red blood cells have increased 2,3 diphosphoglycerate (2,3 DPG) and ATP levels, and improved post transfusion survival and oxygen transport function. This approach has been used to salvage outdated O-positive and O-negative red blood cells. Rejuvenated red blood cells with improved oxygen transport function have been used successfully in anemic patients with and without cardiopulmonary insufficiency in patients undergoing cardiopulmonary bypass and hypothermia, and in instances where non-hemolytic transfusion reactions have been a problem. This new method has been adapted to the preparation of four small aliquots of red blood cells from a single unit for pediatric transfusion.