Peptide fragments of bovine S-antigen, an immunopathogenic retinal autoantigen which mediates experimental autoimmune uveoretinitis, were produced by cyanogen bromide cleavage and used to study antibody-defined epitopes, primarily those defined by antibodies from Lewis rats immunized with the intact antigen or various peptide fragments purified from the digests by HPLC. Antibodies from the sera have been affinity-purified on several of the peptides and examined by western blot analysis and enzyme-linked immunosorbent assay on S-antigen, digests and purified fragments. By immunoblotting it could be shown that five of the purified peptides, CB46, CB47, CB67, CB74 and CB123 were immunogenic, eliciting antibodies which recognized the peptides to which they were prepared; all, except for CB67, elicited antibodies which also bound intact S-antigen. Two more peptides, CB14 and CB27 were not immunogenic and did not contain epitopes. An epitope was also found in CB35, a previously uncharacterized peptide. Using these procedures together with amino acid sequence and composition data, we have been able to determine the origins of the peptides which contain antibody epitopes, including those which we have previously determined to possess epitopes recognized by class II MHC-restricted T cell lines raised to S-antigen and several of the peptides. A T cell line specific for the non-uveitogenic CB47 peptide was unable to transfer the disease.