BACKGROUND: Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage-dependent changes that may affect GSH production. STUDY DESIGN AND METHODS: RBC units (n = 6) in saline-adenine-glucose-mannitol (SAGM) solution were each divided into four transfusion bags and separate treatments were applied: 1) SAGM (control), 2) GSH precursor amino acids, 3) aminoguanidine, and 4) glyoxal. RBCs were sampled during 6 weeks of storage. Rejuvenated RBCs were also analyzed. RESULTS: After 6 weeks, the ATP concentration declined to 50 Â± 5.5% (p < 0.05) of that in the fresh RBCs. For control RBCs, the GSH concentration decreased by 27 Â± 6.5% (p < 0.05) and the rate of GSH synthesis by 45 Â± 8% (p < 0.05). The rate of GSH synthesis in rejuvenated and amino acid-treated RBCs was unchanged after 6 weeks. Modeling identified that the decline in GSH synthesis was due to decreased intracellular substrate concentrations and reduced amino acid transport, secondary to decreased ATP concentration. CONCLUSION: This study has uniquely shown that the glutathione synthesis rate decreased significantly after 6 weeks in stored RBCs. Our results have identified potential opportunities for improvement of banked blood storage.