The loss of cochlear hair cells, or the loss of their capacity to transduce acoustic signals, is believed to be the underlying mechanism in many forms of hearing loss. To develop viral vectors that allow for the introduction of genes directly into the cochleae of adult animals, replication-deficient (E1(-), E3(-)) and replication-defective (E1(-), E3(-), pol(-)) adenovirus vectors were used to transduce the bacterial beta-galactosidase gene into the hair cells of the guinea pig cochlea in vivo. Distortion product otoacoustic emissions, which monitor the functional status of outer hair cells, were measured throughout the viral infection periods to identify hair cell ototoxicity. The results demonstrated that the use of the (E1(-), E3(-)) adenovirus vectors containing CMV-driven LacZ, compromised cochlear function when gradually introduced into scala tympani via an osmotic pump. However, when (E1(-), E3(-), pol(-)) adenoviral vectors containing CMV-driven LacZ were used to transduce cochlear hair cells, there was no loss of cochlear function over the frequency regions tested, and beta-galactosidase (beta-gal) was detected in over 80% of all hair cells. Development of a viral vector that infects cochlear hair cells without virus-induced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for otoprotective strategies to prevent hair cell losses to treat progressive hearing disorders. Moreover, in vivo (E1(-), E3(-), pol(-)) adenovirus mediated gene-transfer techniques applied to adult guinea pig cochleae may be useful in testing several hypotheses concerning what roles specific genes play in normal cochlear function.