We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP^C) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP_23–145, indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP_90–231. Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP_23–145 and PrP_90–231, suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot™, only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.