Recognition of immune-mediated sensorineural deafness that responds to immunosuppressive therapy has led to a search for a diagnostic assay to identify inner ear autoantibodies. Without a confirmed diagnosis of autoimmune disease, many patients have undergone inappropriate immunosuppressive treatment or developed irreversible inner ear damage. Serum from patients with progressive sensorineural hearing loss (n = 54), ulcerative colitis (N = 5), normal controls (N = 14), and animals with experimental autoimmune sensorineural hearing loss (EASNHL) were analyzed by Western blot against fresh bovine inner ear antigen preparations. The hearing loss group (19 [35%]) showed a single-or double-band migrating at 68,000 molecular weight (MW), differing from the normal group (1 of 14 [7%]) which showed a similar band (P = .031). Upon analysis by two-dimensional gel electrophoresis both the EASNHL guinea pigs and a patient reacted against identical components of inner ear antigen. These results suggest an autoimmune basis for disease in patients reacting against the 68,000 MW antigen.