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Background: The Czech Republic decided to build the frozen red blood cell (RBC) bank for military contingency and civil emergency preparedness. Storage methods were validated with (51)Cr recovery measures out to 21 days postthaw., Study Design and Methods: A total of 139 apheresis RBC units collected in additive solution (additive solution 3 [AS-3] or saline-adenine-glucose-mannitol [SAG-M]) were obtained from volunteers by double erythrocytapheresis; all were sterilely glycerolized, frozen, stored for at least 30 days at a minimum of -65 degrees C in collection or cryogenic bags, thawed, sterilely deglycerolized, and stored at 4 +/- 2 degrees C for up to 21 days in AS-3 or SAG-M. Units were sampled before storage and after thaw weekly. Metabolic intermediates and (51)Cr recovery measures were obtained to determine the index of therapeutic effectiveness (ITE)., Results: Despite losses associated with cryoconservation and eventual transfer to cryogenic bags, 3-week storage with optimum ITE was obtained with frozen storage in primary collection or cryogenic bags and postthaw storage in AS-3. Such cells would pass as acceptable in vitro hematology and biochemistry variables., Conclusions: A system for frozen RBC storage with 3-week, postthaw, liquid storage has been validated. With commercially available components and automated processing, it is capable of supporting civilian rare blood and emergency and international military combat and peacekeeping support use patterns.