BACKGROUND Red blood cells (RBCs) are Food and Drug Administration (FDA)-approved for 42-day storage with the use of additive solutions (ASs). However, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) levels in the RBCs decline over this time. These constituents may be restored by treatment with rejuvenation (REJ) solutions. This study was done to assess the response capability of RBCs from 30 to 120 days of storage in three FDA-licensed RBC storage solutions after incubation with a rejuvenating solution of pyruvate, inosine, phosphate, and adenine. METHODS Three units each of RBCs in approved AS (AS-1 [Adsol, Fenwal, Inc.], AS-3 [Nutricel, Medsep Corp.], and AS-5 [Optisol, Terumo Corp.]) were stored under standard conditions at 1 to 6°C for up to 120 days. Aliquots (4 mL) on Days 30, 42, 60, 80, 100, and 120 (± 2 days) were REJ by incubating with Rejuvesol (Encyte Corp.). Control untreated and REJ aliquots were extracted using perchloric acid and stored at -80°C until assayed for 2,3-DPG and ATP. RESULTS RBCs responded to REJ by increasing DPG and ATP contents. The response declined linearly at 0.070 ± 0.008 µmol DPG/g hemoglobin (Hb)/day and 0.035 ± 0.004 µmol ATP/g Hb/day with no differences between ASs. CONCLUSIONS We conclude that Rejuvesol is able to restore ATP and 2,3-DPG levels in RBCs stored up to 120 days in AS. The response diminishes as storage time increases. This rejuvenation (REJ) capability does not seem useful for routine assessment of RBC anabolic capacity in research programs, but may be useful to the investigator when studying unique and novel treatment methods.