It has been shown that sera from patients with autoimmune inner ear disease contain antibodies to several inner ear antigens. We report here the characterization of the 42–43 kDa protein against which a significant number of patients' sera react strongly. After separation of inner ear proteins from guinea‐pig cochleas by SDS‐PAGE, the band corresponding to the 42–43 kDa protein was digested with trypsin and the peptide fragments were separated by high‐performance liquid chromatography. Two fractions were then subjected to amino acid sequencing by the classical automated Edman degradation. The sequence of a stretch of 15 amino acids of the first fragment was identical to that of amino acids 148–162 of β‐actin. The sequence of the 10 amino acids of the second fragment was also identical to β‐actin. On Western blots, monoclonal antibody directed against β‐actin reacted with the inner ear 42–43 kDa proteins. The serum samples from the patients and the monoclonal antibody reacted with the non‐muscle actin used as antigen in Western blotting. Immunoblot analysis of inner ear proteins after two‐dimensional gel electrophoresis showed a spot, corresponding to the region of the 43 kDa as compared to the protein standards. On the basis of these data it is concluded that the target 42–43 kDa protein for antibodies in sera of patients with autoimmune inner ear disease is β‐actin, a molecule, which has important and numerous functions inside cells. This is the first report to identify the cytoskeletal protein β‐actin as a candidate autoantigen in autoimmune inner ear disease.