Human 21-hydroxylase (21-OH) genes containing various mutations, truncations, and deletions were expressed in yeast, and autoantibody binding was studied by Western blotting using patient sera and rabbit antibodies to 21-OH. 21-OH autoantibodies in 13 Addisonian sera showed a marked reduction in their ability to recognize 21-OH mutated at Pro453-->Ser (mean +/- SD, 31 +/- 9% of binding to wild type), whereas the effect on rabbit antibody binding was small (88 +/- 11% of binding to wild type; n = 7). Mutation at Arg339-->His had a less pronounced effect on autoantibody binding (85 +/- 11% of binding to wild type; n = 13) and caused a small enhancement of rabbit antibody binding (124 +/- 16% of binding to wild type; n = 7). These studies indicate that Pro453 has a key role in forming an autoantigenic epitope on 21-OH. It is important to note, however, that the Pro453 mutation caused only partial loss of autoantibody binding, i.e. all Addisonian sera studied still reacted with the mutated protein. This may indicate that each serum sample contains at least two different populations of 21-OH autoantibodies, only one of which recognizes a site dependent on Pro453. A series of more extensive modifications of the 21-OH sequence, including truncations (amino acids 460-494, 448-494, and 418-494) and deletions (amino acids 165-379, 142-240, and 142-280) indicated that most of the sequence of amino acids from 241-494 is important for autoantibody binding. The involvement of such an extensive region of the molecule suggests that the binding sites are generated by three-dimensional folding, with Pro453 having a critical role in forming at least one major autoantigenic epitope.