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We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values).At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at −80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters.Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively.Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3.