Cytotoxic T lymphocytes (CTL) play an important role in the destruction of immunogenic tumors. A novel category of target antigens for CTL concerns normal differentiation antigens as most clearly demonstrated in human melanoma. In the case of B-cell cancers, differentiation antigens normally expressed on B cells may be useful targets. In this report, we have focused on the murine B-cell differentiation antigens CD19 and CD20. We have identified 18 peptide sequences on the basis of major histocompatibility complex (MHC) class-I binding-motifs as candidates for the induction of autoreactive CTL. Six of the peptides were capable of binding efficiently to either Kb or Db and were subsequently used for in vivo induction of CTL. Vaccination with each of three peptides led to peptide-specific CTL. Two peptides were derived from the mCD20 antigen and one from the mCD19 antigen. CTL specific for the mCD19-derived peptide were also capable of killing a syngeneic B-cell tumor line. Recognition of the peptide as well as the tumor cells was shown to be Kb restricted. This is the first report to show that autoreactive CTL recognizing peptides derived from B-cell-specific differentiation antigens can be generated by vaccination with a synthetic peptide.