Congenital heart defects (CHD) are one of the most prevalent birth defects and affect approximately 1 % of neonates born worldwide. Open-heart surgery on cardiopulmonary bypass (CPB) is often required to correct CHD. However, oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) production and antioxidant capacity, has been shown to be a major risk factor during open heart surgery on CPB. The antioxidant  -microglobulin (A1M) has been shown to prevent oxidative stress through its reductase-, heme binding- and radical scavenging abilities. In this study, protein levels of A1M were examined in plasma and urine from neonates during open-heart surgery on CPB as well as the presence of a processed form of A1M, i.e. truncated A1M (t-A1M), in a subgroup of neonates exposed to high concentrations of plasma cell-free hemoglobin. The results obtained in this study indicate that A1M resources are diminished during CPB circulation, followed by significant increases post-operatively. Moreover, when exposed to increased cell-free hemoglobin during CPB, A1M in urine may potentially be cleaved into its truncated form t-A1M. This study provides the basis as to how the antioxidant A1M reacts to the stress that occurs in neonates during CPB circulation.