Cytomegalovirus infects fetuses through the placenta, resulting in various congenital disorders in newborns, including hearing loss. We developed a monoclonal antibody to guinea pig cytomegalovirus (GPCMV) that was available for immunohistochemistry, and investigated the expression of the GPCMV antigen in animal models of direct and congenital infections. Injection of GPCMV, directly to the inner ear, increased the sound pressure level and resulted in labyrinthitis with severe inflammation. Immunohistochemistry detected GPCMV-infected cells mainly in the scala tympani, scala vestibule and spinal ganglion, but rarely in the cochlear duct. Injection of GPCMV to 5-week pregnant guinea pigs resulted in severe labyrinthitis in fetuses. Immunohistochemistry detected GPCMV-infected cells in the perilymph area and spinal ganglion, but not in the endolymph area, including hair cells. These data suggest that the virus spreads via the perilymph and neural routes in the inner ear of both models of direct and congenital infections.