The anti-mitochondrial antibody response in primary biliary cirrhosis (PBC) is primarily directed at E2 components of PDC, OGDC, and BCOADC, and E3BP. Previous work has shown that the immunodominant autoreactive T- cell epitope is the PDC-E2 163-176 peptide, restricted by HLA DR53. To address molecular mimicry and cross-recognition among mitochondrial autoantigens, we analyzed reactivity, including agonism and antagonism assays, to a series of single amino acid–substituted peptides using cloned T-cell lines in PBC and controls. Interestingly, fine specificities were unique for every single T-cell clone, but the clones could be categorized into two distinct groups based on recognition motifs of the T-cell receptor (TCR) ligand: group A 170ExDK173 and group B168EIExD172. 170E is the most critical TCR contact residue for both groups of cloned T-cell lines, whereas 173K and 168E are the critical TCR contact residues for group A and group B cloned T-cell lines, respectively. More importantly, some group A–cloned T-cell lines cross-reacted to human E3BP 34-47, human OGDC-E2 100-113, and several peptides derived from various microbial proteins carrying an ExDK motif, whereas group B–cloned T-cell lines reacted only to E3BP 34-47 carrying an EIExD motif. Furthermore, an RGxG motif was exclusively found in the complementarity-determining region (CDR3) of the TCR Vβ in the group B–cloned T-cell lines, while G, S, and/or R were frequently found in the CDR3 of the TCR Vβ in the group A–cloned T-cell lines. These data provide a framework for understanding molecular mimicry among mitochondrial antigens.