Periventricular leukomalacia (PVL), the major lesion underlying cerebral palsy in survivors of prematurity, is characterized by focal periventricular necrosis and diffuse gliosis of immature cerebral white matter. Causal roles have been ascribed to hypoxiaischemia and maternal-fetal infection, leading to cytokine responses, inflammation, and oligodendrocyte cell death. Because interferon-γ (IFN-γ) is directly toxic to immature oligodendrocytes, we tested the hypothesis that it is expressed in PVL (N = 13) compared to age-adjusted controls (N = 31) using immunocytochemistry. In PVL, IFN-γ immunopositive macrophages were clustered in necrotic foci, and IFN-γ immunopositive reactive astrocytes were present throughout the surrounding white matter (WM). The difference in the number of IFN-γ immunopositive glial cells/high power field (IFN-γ score, Grades 0–3) between PVL cases (age-adjusted mean 2.59 ± 0.25) and controls (age-adjusted mean 1.39 ± 0.16) was significant (p<0.001). In the gliotic WM, the IFN-γ score correlated with markers for lipid peroxidation, but not nitrative stress. A subset of premyelinating (O4+) oligodendrocytes expressed IFN-γ receptors in PVL and control cases, indicating that these cells are vulnerable to IFN-γ toxicity via receptor-mediated interactions. In PVL, IFN-γ produced by macrophages and reactive astrocytes may play a role in cytokine-induced toxicity to premyelinating oligodendrocytes as part of a cytokine response stimulated by ischemia and/or infection.