Aims: Transfusion with stored red blood cells (RBCs) is associated with increased morbidity and mortality. Peroxiredoxin-2 (Prx-2) is a primary RBC antioxidant that limits hydrogen peroxide (H2O2)-mediated toxicity. Whether Prx-2 activity is altered during RBC storage is not known. Results: Basal and H2O2-induced Prx-2 activity was measured in RBCs (stored for 7-35 days). Basal Prx-2 thiol oxidation increased with RBC age, whereas H2O2-dependent formation of dimeric Prx-2 was similar. However, reduction of Prx-2 dimers to monomers became progressively slower with RBC storage, which was associated with increased H2O2-induced hemolysis. Surprisingly, no change in the NADPH-dependent thioredoxin (Trx)/Trx-reductase system, which recycles dimeric Prx-2, was observed in stored RBCs. Using mouse RBCs expressing human wild type (β93Cys) or hemoglobin (Hb) in which the conserved β93Cys residue is replaced by Ala (β93Ala), a role for this thiol in modulating Prx-2 reduction was demonstrated. Specifically, Prx-2 recycling was blunted in β93Ala RBC, which was reversed by carbon monoxide-treatment, suggesting that heme autoxidation-derived H2O2 maintains Prx-2 in the oxidized form in these cells. Moreover, assessment of the oxidative state of the β93Cys in RBCs during storage showed that while it remained reduced on intraerythrocytic Hb in stored RBC, it was oxidized to dehydroalanine on hemolyzed or extracellular Hb. Innovation: A novel mechanism for regulated Prx-2 activity in RBC via the β93Cys residue is suggested. Conclusion: These data highlight the potential for slower Prx-2 recycling and β93Cys oxidation in modulating storage-dependent damage of RBCs and in mediating post-transfusion toxicity. Antioxid. Redox Signal. 22, 294-307.