The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrP(Sc)), is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrP(Sc) accumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrP(Sc) formation reduced PrP(Sc) accumulation in cells persistently infected with prions. The 50% effective dose was as low as approximately 1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrP(C)) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb-PrP(C) complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrP(Sc) formation by interfering with the regular PrP(C) degradation pathway.