The polyclonal nature of antimitochondrial autoantibodies and the limited success of generating human monoclonal antibodies have made analysis of fine specificity and antibody heterogeneity difficult to define. The major autoantigen of primary biliary cirrhosis is the E2 component of the pyruvate dehydrogenase pathway (PDC-E2). To address the relative importance of the region(s) in the PDC-E2 inner lipoyl domain to antibody binding, we report herein detailed profiles of 12 PDC-E2—specific antigen-binding fragments, SP1 through SP12, derived by screening of a combinatorial immunoglobulin library (derived from a primary biliary cirrhosis patient) with full-length native PDC-E2. All antigen-binding fragments are IgG isotypes and include a similar number of λ- and κ-chains. The antigen-binding fragments react specifically to PDC-E2 with high affinity (Ka = 10−7 to 10−10 mol/L−1) and recognize a conformational epitope in the inner lipoyl domain of PDC-E2. Furthermore, the antibodies demonstrate substantial heterogeneity in recognition of different recombinant PDC-E2 fragments and differential recognition patterns against mutant constructs of the human PDC-E2 inner lipoyl domain (amino acid residues 91 to 227). In addition, five of the antigen-binding fragment clones (SP1, 3, 4, 8 and 12) demonstrate different staining patterns on biliary epithelial cells of patients with primary biliary cirrhosis but not control liver disease; some antigenbinding fragments specifically stained the apical region of biliary epithelium, a pattern distinct from that of typical mitochondrial staining. The response to the inner lipoyl domain is not, however, monospecific, and there is much more heterogeneity in fine specificity than could be accounted for by arbitrary reshuffling of variable immunoglobulin heavy and light chains into unnatural combinations. (HEPATOLOGY 1994;20:574-;583).