Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform (PrP(Sc)) of a normal cellular protein (PrP(C)). Shared sequence identity of PrP(Sc) with PrP(C) has limited the detection sensitivity of immunochemical assays, as antibodies specific for the disease-causing PrP(Sc) isoform have not been developed. Here we report the generation of three new monoclonal antibodies (MAbs) to PrP, which were isolated following immunization of Prnp(0/0) Balb/cJ mice with highly purified PrP(Sc) isolated from brain lipid rafts. Epitope mapping using synthetic PrP peptides revealed that the three MAbs bind different epitopes of PrP. The DRM1-31 MAb has a conformational epitope at the proposed binding site for the putative prion conversion co-factor "protein X." The DRM1-60 MAb binds a single linear epitope localized to the β2-α2 loop region of PrP, whereas DRM2-118 binds an epitope that includes sequences within the octarepeat region and near the site of N-terminal truncation of PrP(Sc) by proteinase K. Our novel anti-PrP MAbs with defined PrP epitopes may be useful in deciphering the conformational conversion of PrP(C) into PrP(Sc).