Sera from patients with idiopathic Addison's disease commonly react with the zona glomerulosa of adrenal cortex. We used high-resolution western blot analysis of an adrenal microsomal fraction to investigate the target of these antibodies. A protein with an apparent molecular weight of 54 kDa was recognised as the common and major component. Sera identifying this autoantigen (from 12 of 16 patients) also showed strong immunofluorescence staining of a steroid-producing human adrenal adrenocortical cell line, NCI-H295. On application of antisera specific for different cytochrome P450 steroidogenic enzymes (sidechain cleavage enzyme, 21-hydroxylase, 17α-hydroxylase, 11β-hydroxylase) the mobility of the 54 kDa protein in western blots corresponded to that of 21-hydroxylase. This parallel behaviour was confirmed by immunoprecipitation, electrophoresis, and autoradiography with the various sera and ³⁵S-methionine-labelled NCI-H295 cell lysates. Preabsorptions of ³⁵S-methionine-Labelled cell lysates with the antiserum to 21-hydroxylase, but not with the other enzyme antisera, abolished precipitation of the 54 kDa autoantigen with the patient sera. These results indicate that 21-hydroxylase (P450c21), prominent in the zona glomerulosa of the adrenal cortex, is a major autoantigen in idiopathic Addison's disease.