The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP^C) into PrP^Sc, a partially protease-resistant and infectious conformer. However, the mechanism by which PrP^Sc causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP^C, were found to be reduced in the brains of mice infected with the RML strain of prions , implying that Sho levels may reflect the presence of PrP^Sc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP^Sc. Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP^Sc. Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP^Sc during prion disease.