A quantitative assay for human adrenal autoantibodies has been developed to aid in the detection and isolation of human adrenal antigens. To define the subcellular location (s) of the antigen (s) capable of binding with these antibodies, we have quantitated both antibody binding to various adrenal subcellular fractions and the adrenal autoantibody binding inhibition caused by each subcellular fraction. To further define the subcellular location of the autoantibody binding, each fraction was assayed for organelle-specific marker enzyme activities. Enzyme activities were correlated to adrenal autoantibody binding to each fraction by linear regression. Of the materials tested, both antibody binding and inhibition of binding were most highly correlated with adrenal subcellular fractions enriched with cytochrome-c reductase and 5’-nucleotidase (r = 0.98; P < 0.05). Thus, our data support the localization of adrenal autoantigen(s) in the microsomes, plasma membrane, or both. © 1990 by The Endocrine Society.