Adrenal autoantibodies characteristic of autoimmune Addison disease are directed towards steroid 21-hydroxylase (21-OH; EC 1.14.99.10). We describe a new assay to measure 21-OH autoantibodies (21-OH Abs), based on immunoprecipitation by the antibodies of 35S-labeled human 21-OH. Using this immunoprecipitation assay (IPA), we detected 21-OH Abs in 42 of 64 (66%) patients with Addison disease and in 14 of 19 (74%) patients with autoimmune polyendocrine syndromes type I and type II. No 21-OH Abs were detected by the IPA in any patients with Addison disease attributable to tuberculosis (n = 9) or adrenoleukodystrophy (n = 9) or in patients with autoimmune thyroid disease (n = 28), systemic lupus erythematosus (n = 10), myasthenia gravis (n = 10), rheumatoid arthritis (n = 10), or insulin-dependent diabetes mellitus (n = 12). None of the 26 sera from healthy normal blood donors was positive for 21-OH Abs by the assay. We found good agreement between 21-OH Abs measured by IPA and by Western blotting (r = 0.83, n = 123, P < 0.001). The inter- and intraassay CVs for IPA were well < 10% at high, medium, and low concentrations of 21-OH Abs. Overall, our studies indicate that the IPA provides a specific, sensitive, and convenient system for measuring 21-OH Abs.