OBJECTIVE: Hemolysis, occurring during cardiopulmonary bypass, is associated with lipid peroxidation and postoperative acute kidney injury. Acetaminophen inhibits lipid peroxidation catalyzed by hemeproteins and in an animal model attenuated rhabdomyolysis-induced acute kidney injury. This pilot study tests the hypothesis that acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass. DESIGN: Single-center prospective randomized double-blinded study. SETTING: University-affiliated pediatric hospital. PATIENTS: Thirty children undergoing elective surgical correction of a congenital heart defect. INTERVENTIONS: Patients were randomized to acetaminophen (OFIRMEV [acetaminophen] injection; Cadence Pharmaceuticals, San Diego, CA) or placebo every 6 hours for four doses starting before the onset of cardiopulmonary bypass. MEASUREMENT AND MAIN RESULTS: Markers of hemolysis, lipid peroxidation (isofurans and F2-isoprostanes), and acute kidney injury were measured throughout the perioperative period. Cardiopulmonary bypass was associated with a significant increase in free hemoglobin (from a prebypass level of 9.8 ± 6.2 mg/dL to a peak of 201.5 ± 42.6 mg/dL postbypass). Plasma and urine isofuran and F2-isoprostane concentrations increased significantly during surgery. The magnitude of increase in plasma isofurans was greater than the magnitude in increase in plasma F2-isoprostanes. Acetaminophen attenuated the increase in plasma isofurans compared with placebo (p = 0.02 for effect of study drug). There was no significant effect of acetaminophen on plasma F2-isoprostanes or urinary makers of lipid peroxidation. Acetaminophen did not affect postoperative creatinine, urinary neutrophil gelatinase-associated lipocalin, or prevalence of acute kidney injury. CONCLUSION: Cardiopulmonary bypass in children is associated with hemolysis and lipid peroxidation. Acetaminophen attenuated the increase in plasma isofuran concentrations. Future studies are needed to establish whether other therapies that attenuate or prevent the effects of free hemoglobin result in more effective inhibition of lipid peroxidation in patients undergoing cardiopulmonary bypass. Copyright © 2014 by the Society of Critical Care Medicine and the World.