The best method of monitoring anticoagulation during extracorporeal membrane oxygenation (ECMO) is unknown. We conducted a prospective observational study in a tertiary pediatric intensive care unit. Antifactor Xa, antithrombin (AT), and factor VIII activity (FVIII) were measured in blood samples collected at 6, 12, and every 24 hours, respectively, of ECMO. We enrolled 34 children who underwent 35 ECMO runs from April 2008 to September 2010. Activated clotting time (ACT) and heparin doses were higher, whereas antifactor Xa levels were lower in neonates compared to infants/children. Median antifactor Xa was 0.4 IU/ml, median AT was 60%, and median FVIII was 67%. Heparin infusion rate, antifactor Xa, and antithrombin (AT) increased, FVIII was stable, and ACT decreased with each day on ECMO. ACT had poor agreement with antifactor Xa (42%). AT was inversely correlated with ACT (r = -0.33), even after adjusting for heparin dose, and positively correlated with antifactor Xa (r = 0.57). This study emphasizes the age differences as well as the variability over days of coagulation monitoring assays during ECMO. ACT is poorly correlated with antifactor Xa and AT modifies the relationship between ACT and the heparin dose, indicating that results should be interpreted with caution when managing anticoagulation on ECMO. Additional studies are warranted to determine optimal ECMO anticoagulation monitoring.