Recent studies have demonstrated that delivery of genes to the inner ear can achieve a variety of effects ranging from support of auditory neuron survival to protection and restoration of hair cells, demonstrating the utility of vector based gene delivery. Translation of these findings to useful experimental systems or even clinical applications requires a detailed understanding of the pharmacokinetics of gene delivery in the inner ear. Ideal gene delivery systems will employ a well tolerated vector which efficiently transduces the appropriate target cells within a tissue, but spare non-target structures. Adenovectors based on serotype 5 (Ad 5) are commonly used vectors, are easy to construct and have a long track record of efficacious gene transfer in the inner ear. In this study we demonstrate that distribution of Ad5 vector occurs in a basal to apical gradient with rapid distribution of vector to the vestibule after delivery via a round window cochleostomy. Transduction of the vector and expression of the delivered transgene occurs by 10 min post vector delivery. At 24 h post delivery only 16% of vector that was initially detectable within the inner ear by quantitative PCR remained. Perilymph sampling was used to determine that vector concentrations in perilymph peaked at 30 min post delivery and then declined rapidly. Understanding these basic distribution patterns and parameters for delivery are important for the design of gene delivery vectors and vital for modeling dose responses to achieve safe efficacious delivery of a therapeutic agent.