Hypoglycemia is associated with gray and white matter injury in immature brain, but the specific mechanisms responsible for hypoglycemic brain injury remain poorly defined. We postulated that mitochondrial electron transport chain function is altered during hypoglycemia due to the decreased availability of reducing equivalents, and that altered activity of the electron transport chain would increase mitochondrial production of free radicals and lead to mitochondrial oxidant injury. The present study tests the hypothesis that production of reactive oxygen species (ROS) by cerebral mitochondria is increased during acute hypoglycemia. Studies were performed in an awake, chronically catheterized newborn piglet model. Hypoglycemia (blood glucose 1 mmol/L for 2 h) was induced using a bolus of intravenous lispro insulin, 25 U/kg. Superoxide and hydrogen peroxide production by mitochondria isolated from cerebral cortex of normoglycemic and hypoglycemic newborn piglets was measured using lucigenin- and luminol-derived chemiluminescence. After 2 h of hypoglycemia, superoxide generation was 60% higher and hydrogen peroxide generation was two-fold higher in mitochondria from hypoglycemia animals than in controls (p < 0.005). These data confirm that the ability of the mitochondria to produce ROS is increased after hypoglycemia in immature brain, and are, to our knowledge, the first evidence that ROS may play a role in brain injury due to neonatal hypoglycemia. Increased mitochondrial ROS production could result in alterations in brain structure and function due to oxidant injury to mitochondrial proteins and DNA or changes in oxidant-sensitive signal transduction pathways in brain.