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Objective To study expression of adenovira1-mediated Hathl-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane (RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with AdHath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and fro zensections were examined. Results ABR tests showed no significant change of hearing after the surgery.Strong fluorescence staining in the cochleae was seen in Ad-Hathl-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with tittle decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.