Growing evidence has implicated the involvement of autoreactive T lymphocytes in the pathogenesis of primary biliary cirrhosis (PBC). We have recently taken advantage of motif prediction analysis of HLA‐A*0201 and identified the first major histocompatibility complex (MHC) class I restricted epitope, amino acids 159 to 167 on E2 components of pyruvate dehydrogenase complexes (PDC‐E2), the major mitochondrial antigens in PBC. The mechanisms involved in the selection of epitope peptide(s) that comprise the PDC‐E2–specific autoreactive cytotoxic T lymphocytes (CTLs) are unknown and likely involve other epitopes on PDC‐E2 restricted by MHC class I molecules. To address this issue, a comprehensive mapping of the CTL epitope repertoire on the PDC‐E2 molecule that binds HLA‐A*0201 was performed to provide further clues regarding the role of CTLs. We used the T2 cell line to screen 79 overlapping 15mer peptides, spanning the entire PDC‐E2 molecule. Six of the 79 peptides exhibited significantly higher binding activity to HLA‐A*0201 than the other 15mer peptides. Two of these 6 peptides induced CTL lines from patients with PBC. Fine mapping with N‐terminus or C‐terminus truncated peptides identified 10mer peptide, PDC‐E2 amino acids 165 to 174, which is a novel CD8 epitope restricted by HLA‐A*0201. In conclusion, using a combination of the 15mer peptide library screening with the T2 binding assay and also the induction of CTL lines with candidate peptides, we have defined a novel HLA‐A*0201–restricted epitope PDC‐E2 165 to 174 in patients with PBC. These data will become important in the development of altered peptide ligands to modulate disease activity.