Abstract Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnpo/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnpo/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnpo/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnpo/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnpo/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.