Background: Cell-free hemoglobin (Hb) forms in stored red blood cells (RBCs) as a result of hemolysis. Studies suggest that this cell-free Hb may decrease nitric oxide (NO) bioavailability, potentially leading to endothelial dysfunction, vascular injury, and multiorgan dysfunction after transfusion. We tested the hypothesis that moderate doses of stored RBC transfusions increase cell-free Hb and decrease NO availability in postoperative surgical patients. Methods: Twenty-six patients undergoing multilevel spine fusion surgery were studied. We compared those who received no stored RBCs (n = 9) with those who received moderate amounts (6.1 ± 3.0 units) of stored RBCs over 3 perioperative days (n = 17). Percent hemolysis (cell-free Hb), RBC-NO (heme-NO), and plasma nitrite and nitrate were measured in samples from the stored RBC bags and from patients' blood, before and after surgery. RESULTS: Posttransfusion hemolysis was increased approximately 3.5-fold over preoperative levels (P = 0.0002) in blood samples collected immediately after surgery but not on postoperative days 1 to 3. Decreases in both heme-NO (by approximately 50%) and plasma nitrite (by approximately 40%) occurred postoperatively, both in nontransfused patients (P = 0.036 and P = 0.026, respectively) and transfused patients (P = 0.0068 and P = 0.003, respectively) and returned to preoperative baseline levels by postoperative day 2 or 3. Postoperative plasma nitrite and nitrate were decreased significantly in both groups, and this change was slower to return to baseline in the transfused patients, suggesting that blood loss and hemodilution from crystalloid administration contribute to this finding. CONCLUSIONS: The decrease in NO metabolites occurred irrespective of stored RBC transfusions, suggesting this decrease may be related to blood loss during surgery and hemodilution rather than to scavenging of NO or inhibition of NO synthesis by stored RBC transfusions.