Patients with idiopathic Addison's disease are characterized by cytoplasmic adrenal autoantibodies, detectable by indirect immunofluorescence of cryocut sections of human adrenal cortex. Recently, autoantibodies that bind a 55-kilodalton protein in the microsomal fraction of adrenal gland extracts identified to be the cytochrome P450 enzyme 21-hydroxylase have been found in Addisonian patient sera. We confirm the finding and report here the autoantigenic epitopes involved in the autoantibody reactivity using recombinant DNA technology. Six cDNA fragments spanning different regions of the 21-hydroxylase gene were expressed as fusion proteins with glutathione S-transferase in Escherichia coli. Immunoblot analyses were used to evaluate the reactivity of the recombinant proteins with patients' sera to determine the autoepitopes involved. We found that a conserved region (amino acids 164-356) reacted with 25 of 30 adrenal autoantibody-positive sera tested. One serum sample reacted only with the amino portion of the 21-hydroxylase (amino acids 1-162). In addition, 4 other enzymes important to steroid hormone biosynthesis, 11 beta-hydroxylase, 17 alpha-hydroxylase, side-chain cleavage enzyme P450, and 3 beta-hydroxysteroid dehydrogenase, were expressed in E. coli, but none of them gave positive autoantibody reactions by Western blot assays, even using sera from 5 patients with type I autoimmune polyglandular syndrome. The availability of recombinant antigens has permitted structural analysis of the autoepitopes involved in the autoimmune response to 21-hydroxylase in Addison's disease. Our findings should lead to the development of a simple and specific tool for immunodiagnosis of the disease.