Background: Although previous studies have identified an association between the transfusion of relatively older red blood cells (RBCs) (storage ≥14 days) and adverse outcomes, they are difficult to interpret because the majority of patients received a combination of old and fresh RBC units. To overcome this limitation, we compared in-hospital mortality among patients who received exclusively old versus fresh RBC units during the first 24 hours of hospitalization. Methods: Patients admitted to a Level I trauma center between January 2000 and May 2009 who received ≥1 unit of exclusively old (≥14 days) vs. fresh (<14 days) RBCs during the first 24 hours of hospitalization were identified. Risk ratios (RRs) and 95% confidence intervals (CIs) were calculated for the association between mortality and RBC age, adjusted for patient age, Injury Severity Score, gender, receipt of fresh frozen plasma or platelets, RBC volume, brain injury, and injury mechanism (blunt or penetrating). Results: One thousand six hundred forty-seven patients met the study inclusion criteria. Among patients who were transfused 1 or 2 RBC units, no difference in mortality with respect to RBC age was identified (adjusted RR, 0.97; 95% CI, 0.72-1.32). Among patients who were transfused 3 or more RBC units, receipt of old versus fresh RBCs was associated with a significantly increased risk of mortality, with an adjusted RR of 1.57 (95% CI, 1.14-2.15). No difference was observed concerning the mean number of old versus fresh units transfused to patients who received 3 or more units (6.05 vs. 5.47, respectively; p = 0.11). Conclusion: In trauma patients undergoing transfusion of 3 or more RBC units within 24 hour of hospital arrival, receipt of relatively older blood was associated with a significantly increased mortality risk. Reservation of relatively fresh RBC units for the acutely injured may be advisable. Copyright © 2010 by Lippincott Williams & Wilkins.