Gene transfer into the cells of the cochlea is useful for both research and therapy. Bovine adeno-associated virus (BAAV) is a new viral vector with potential for long-term gene expression with little or no side effects. In this study, we assessed transgene expression using BAAV with beta-actin-GFP as a reporter gene, in the cochleae of normal and deafened guinea pigs. We used two different routes to inoculate the cochlea: scala media (SM) or scala tympani (ST). Auditory brainstem response assessments were carried out before inoculation, 7 days after inoculation and immediately before killing, to assess the functional consequences of the treatment. We observed threshold shifts because of the surgical invasion, but no apparent pathology associated with the virus. Fourteen days after the injection, animals were killed and cochleae assessed histologically. Epi-fluorescence showed that BAAV transduced the supporting cells of both normal and deafened animals through SM and ST inoculations. Transgene expression in cells of the membranous labyrinth after ST inoculation is an important outcome because of the greater feasibility of this route for future clinical application. BAAV facilitates efficient transduction of the membranous labyrinth epithelium with minimum pathogenicity and may become clinically applicable for inner ear gene therapy.