Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a beta-galactosidase (beta-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of beta-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of beta-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of beta-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express beta-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.