Autoantibodies against 21-hydroxylase are common in idiopathic Addison's disease. The objective of this study was to determine the frequency of these antibodies in different endocrine autoimmune diseases with or without adrenal insufficiency and to follow up their evolution. We have used a radiobinding assay based on 125-I labeled 21-hydroxylase (21-OH-AB, RSR, UK). Serum samples with 21-hydroxylase antibodies levels equal or greater than 1.0 U/ml were considered as positive. We found these autoantibodies in 21/23 idiopathic Addisonian patients, in 0/18 patients with isolated hypothyroïdism, in 0/6 patients with isolated Grave's disease, in 2/14 patients with isolated ovarian failure, in 1/27 patients with 2 ou more associated autoimmune diseases without adrenal insufficiency. The comparison between patients with or without adrenal insufficiency has shown, for this assay, a sensibility of 91 %, a specificity of 96 %. The 21-hydroxylase autoantibodies were followed up in 4 patients with Addison's disease and showed progressive decreasing levels. We can not exclude that the addisonian sera might become negative for these antibodies after the total destruction of adrenal cortex. In conclusion, the presence of 21-hydroxylase autoantibodies is highly specific for idiopathic Addison's disease and the level of these antibodies decrease with disease duration.