In vivo gene transfer in a large group of RPE65 null mutation dogs have been recently performed. The present study was aimed at determining, through visual behavioral and electroretinographic (ERG) testing, if there is a volume effect of the gene construct administered. Eleven Beagle-Briard dogs homozygous for the RPE65 null mutation and two unaffected control dogs were included. Affected animals were unilaterally treated with either a high (70–100 l; N = 6) or a low volume (30–60 l; N = 5) of subretinally injected rAAV.RPE65 construct, at the age of 4 months to 2.5 years. Fellow eyes were treated with a subretinal injection of rAAV.GFP or sham operated and used as internal controls. Retinal function was measured pre- and 10–12 weeks post-surgically, using simultaneous bilateral full-field flash ERGs. A significant improvement in all ERG responses studied was identified for the high volume treated group compared to pre-surgical parameters. A significant improvement for the high intensity scotopic a-wave response for the low volume rAAV.RPE65 treated group was also found. Objective and subjective dim and day light visual maze testing, in eight of the affected treated animals, and the two control dogs, revealed better vision in daylight than in dim light for all animals. Vision in dogs treated with the high volume of gene construct was significantly better in day light than in dim light. No significant difference was noted between day and dim light testing for the control group or those animals treated with a low volume of the gene construct. Significantly better vision was noted in the control group when compared with the low volume group under dim light conditions, and the high volume group under day light conditions. No significant difference in functional vision could be identified between the high volume treated animals and control animals in day light conditions. These findings support the hypothesis that functional vision is improved by subretinal rAAV.RPE65 injection in a volume-dependent manner.