Scientific investigations utilising human explants obtained from eye cups are uncommon due to methodological difficulties, inconsistent tissue characterisation and technical challenges in experimentation. In this study it was hypothesised that human explants containing intact retinal-pigment epithelium (RPE), choroid and sclera (RCS explants) could be used as a suitable model for AAV mediated gene therapy studies. RCS explants were dissected from donor tissue provided by the Queensland Eye Bank and transduced overnight using AAV2, AAV5, AAV6 or AAV8 with 1-3 ×1010 vector copy genomes expressing secreted luciferase. RCS explants were efficiently transduced using AAV2, AAV5, AAV6 and AAV8 vectors. AAV5 and AAV6 were significantly more efficient in transducing RCS explant superstructure than AAV2 and AAV8 (P<0.05). When a 1:2 dilution of vitreous humour was added, AAV2 and AAV6 vector transduction was significantly inhibited (P<0.05), however, both AAV5 and AAV6 transduction remained significantly more efficient than AAV2 and AAV8 in these conditions (P<0.05). RCS explants could be kept in culture for a minimum of 21 days without significant change in luciferase expression. Transduction efficiencies from the intact explant were different to those obtained using isolated primary mixed RPE/choroidal culture from the same source, in which AAV2 was the most efficient vector in transducing cells obtained from RCS explants, suggesting cell-cell interactions and barriers are important in AAV transduction of the outer eye structures. These findings suggest human RCS explants are a robust, useful and highly externally valid model for investigating gene therapy of the RPE and choroid.