Immunological mechanisms are thought to play an important role in the pathogenesis of some cochleo-vestibular diseases. This study attempts to present further evidence of autoantibodies reactive against guinea pig inner ear proteins found in patients with autoimmune inner ear diseases (AIED) and specifically identifies the main target antigens of these antibodies. Sera from 110 patients with a clinical diagnosis of either rapidly progressive sensorineural hearing loss (n = 32). Ménière's disease (n = 41), sudden deafness (n = 6) or other aetiologies of hearing loss (n = 11) were screened by the Western blot technique. Forty-four percent of the patients' sera had antibodies to several inner ear proteins, of which the 30, 42 and 68 kDa proteins were found to be the most reactive. These highly reactive proteins were identified by gas-phase micro sequencing after digestion with trypsin and separation of peptide fragments by high-performance liquid chromatography. A partial sequence of each protein was determined. These data, together with those obtained from 2-dimensional gel electrophoresis followed by Western blotting, demonstrated that the 30 and 42 kDa inner ear proteins are the major peripheral myelin protein P0 and the beta-actin protein, respectively, while sequence analysis indicated that the 68 kDa protein is novel. These findings further support the hypothesis that several populations of antibodies may contribute to the enhanced immunological activity of AIED patients. They also add a new dimension to our knowledge of AIED and may open new avenues in the development of simple serological assays, which are easier to perform and more rapid than Western blotting.