Autoantibodies to the pyruvate dehydrogenase complex (PDC) are present in the serum of more than 95% of patients with primary biliary cirrhosis (PBC), the major epitope being the inner lipoyl domain of the E2 component. Immunoblotting suggests a similar prevalence of antibodies to a tightly associated lipoic acid–containing protein, E3 binding protein (;E3BP). Attempts to resolve E3BP from E2 have been unsuccessful, restricting study of the nature and significance of antibody responses to the individual proteins. In particular, it is unclear (;1) whether there is true cross-reactivity between E3BP and E2 and, if so, which is the originating response and (;2) whether autoantibodies preferentially bind a lipoylated epitope on E3BP as is the case with PDC-E2. In this study, complementary DNAs encoding rE2, full-length rE3BP, its single lipoyl domain (;rLip), and core domain (;rE3BPCore) were cloned, and the proteins were expressed in Escherichia coli. Sera from 47 PBC patients were studied by immunoblotting and enzyme-linked immunosorbent assay (;ELISA) against rE2, rE3BP, rE3BPCore, and both unlipoylated (;U) and lipoylated (;L) rLip. All sera were reactive by ELISA to some degree with all recombinant proteins except rE3BPCore, to which only 6 of 47 showed any reactivity. Significant correlations (;P < .0001) were observed when comparing absorbance values for rE3BP with both rLip (;U) (;r = 0.793) and (;L) (;r = 0.963). The mean absorbance for rLip (;U, 0.26 ± 0.05) was, however, significantly lower than the absorbance for rLip (;L) (;0.78 ± 0.12; P < .0001). After probing by immunoblotting and elution of antibodies from rE2 and rE3BP, subsequent reprobing against the components in whole PDC revealed true cross-reactivity. In summary, the response to E3BP is primarily directed against the lipoylated domain of the protein. It still remains unclear, however, whether the initial breakdown of tolerance is to E2 or E3BP.