Oxidative damage is implied in perinatal hypoxic-ischemic brain injury, most importantly in white matter. Nonprotein-bound iron (NPBI) catalyzes the formation of toxic hydroxyl radicals. We measured the extracellular level of NPBI through microdialysis in the cortex, striatum, and periventricular white matter before, during and after severe hypoxia in newborn pigs. NPBI was analyzed by a new spectrophotometric method in which ferrous iron is chelated by bathophenanthroline. NPBI was present in all brain areas under baseline conditions and increased in white matter from 0.97 (0.69) to 2.75 (1.85) µmol/l (not corrected for recovery rate) during early reoxygenation. NPBI may contribute to oxidative injury after perinatal hypoxic insults.