Neutral endopeptidase (NEP) is the first target antigen identified on podocytes in human membranous nephropathy (MN). Cytotoxic T lymphocytes (CTLs) are considered essential for glomerular destruction in MN model. The aim of this study was to show that the CTL epitopes of NEP could be used to design more effective and better tolerated therapies. The CTL epitopes of NEP were screened using the long-distance prediction system SYFPEITHI and the Bioinformatics and Molecular Analysis Section of the MHC Peptide Binding Predictions program. Peptides were synthesized and immunoreactivity was assessed by peptide-MHC-binding affinity assay, cytotoxicity assay and HLA-A2.1/Kb transgenic mice immunization. Five candidates were identified according to the high scores generated by the computer predicting system. Peptide NEP(375-383) (FIMDLVSSL), which up-regulated HLA-A2.1 molecular expression, showed a high affinity to HLA-A2.1, whereas NEP(268-276), NEP(297--305) and NEP(492-500) (QLALEMNKV, MLLYNKMRL and KLNNEYLEL) showed a moderate affinity and NEP(559-567) (ILQPPFFSA) only had a low affinity. Cytotoxicity assay further showed that NEP(268-276) and NEP(375-383) could induce NEP-specific CTL responses in vitro. Unexpectedly, we found that a single CTL epitope, NEP(375-383), could induce proteinuria and glomerular injury in HLA-A2.1/K(b) transgenic mice in vivo. HLA-A*0201-restricted CTL epitope NEP(375-383) can serve as a potential candidate for designing MN vaccine.