The search for a diagnostic assay in patients with autoimmune inner ear disease has led to the preparation of antigens from the inner ear, a presumed target in this disorder. In order to standardize the antigen preparations currently being used in the Western blot immunoassay, we have examined several distinct extraction procedures that employ well-known solutions and detergents. Results of this investigation clearly show that antigens of interest (68 kd, 33 to 35 kd, and 32 kd) are optimally extracted with a detergent (0.5% sodium dodecyl sulfate) and that bovine serum albumin, a potential contaminant, can be removed in the water-soluble fractions. Purification of inner ear antigens by these methods will lead to more reproducible results in immunoblotting, as well as a greater opportunity to identify the mechanisms involved in autoimmune inner ear disease.