There are a number of genetic diseases that affect the cochlea early in life, which require normal gene transfer in the early developmental stage to prevent deafness. The delivery of adenovirus (AdV) and adeno-associated virus (AAV) was investigated to elucidate the efficiency and cellular specificity of transgene expression in the neonatal mouse cochlea. The extent of AdV transfection is comparable to that obtained with adult mice. AAV-directed gene transfer after injection into the scala media through a cochleostomy showed transgene expression in the supporting cells, inner hair cells (IHCs), and lateral wall with resulting hearing loss. On the other hand, gene expression was observed in Deiters cells, IHCs, and lateral wall without hearing loss after the application of AAV into the scala tympani through the round window. These findings indicate that injection of AAV into the scala tympani of the neonatal mouse cochlea therefore has the potential to efficiently and noninvasively introduce transgenes to the cochlear supporting cells, and this modality is thus considered to be a promising strategy to prevent hereditary prelingual deafness.