Periventricular leukomalacia (PVL), the major substrate of cerebral palsy in survivors of prematurity, is defined as focal periventricular necrosis and diffuse gliosis in immature cerebral white matter. We propose that nitrosative and/or oxidative stress to premyelinating oligodendrocytes complicating cerebral ischemia in the sick premature infant is a key mechanism of injury interfering with maturation of these cells to myelin-producing oligodendrocytes and subsequent myelination. Using immunocytochemical markers in autopsy brain tissue from 17 PVL cases and 28 non-PVL controls, we found in the PVL cases: 1) selective regionalization of white matter injury, including preferential involvement of the deep compared to intragyral white matter; 2) prominent activation of microglia diffusely throughout the white matter; 3) protein nitration and lipid peroxidation in premyelinating oligodendrocytes in the diffuse component; 4) preferential death of premyelinating oligodendrocytes diffusely; and 5) virtual sparing of the overlying cerebral cortex, as demonstrated by markers of activated astrocytes and microglia. These data establish that PVL is primarily a white matter disease that involves injury to premyelinating oligodendrocytes, potentially through activation of microglia and release of reactive oxygen and nitrogen species. Agents that prevent nitrosative and oxidative stress may play a key role in ameliorating PVL in premature infants in the intensive care nursery.