Scrapie-infected mice are considered a model for study in prion diseases, which are characterized by the progressive accumulation in the brain of an abnormal isoform (PrPsc) of the normal cellular prion protein (PrPc). Increasing data suggest that the neurodegenerative process in prion diseases may result, at least partially, from a defect in antioxidant function, but so far in vivo oxidative stress remains poorly documented. We report here that 4-hydroxynonenal, a lipid peroxidation by-product, forms protein adducts in brains of scrapie-infected mice and of Creutzfeldt-Jakob disease affected patients. In scrapie mice, studies on the progression of PrPsc accumulation, glial activation, ubiquitin deposition, and 4-HNE adduct formation allowed us to conclude the late occurrence of oxidative damage in the course of the disease. Massive 4-HNE accumulation was identified in astrocytes, but not in neurons or microglial cells. These findings suggest an important oxidative stress (and subsequent lipid peroxidation) in astrocytes, with possible consequences on their neuronal trophic function.