cDNA clones for bovine retinal S-antigen (48K protein) have been isolated and sequenced. Secondary structure prediction for the deduced amino acid sequence suggest that the protein has predominantly beta-conformation with a helical region at the C-terminus. Local regions of sequence similarity with alpha-transducin have been observed, including the sites subject to ADP-ribosylation by pertussis and cholera toxins. Thirty-nine tryptic peptides and ten CNBr peptides of native S-antigen were purified and partially sequenced, showing good agreement with the sequence deduced from the cDNA analysis. The locations of two monoclonal epitopes have been determined by different methods. Furthermore, one synthetic peptide corresponding to the determined amino acid sequence was found to be highly pathogenic for the induction of experimental autoimmune uveitis (EAU). Native S-antigen has further been shown to be glycosylated, which has implications for its antigenicity.