Introduction: AAV2 delivery of the RPE65 cDNA to the retina of blind RPE65-deficient dogs or rd12 mice restores vision as determined electrophysiologically and behaviorally. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis, but safety and dose-efficacy within a non-toxic range with this vector have not been defined. Here we report the results of toxicology and biodistribution studies in two mammalian species, dogs and rats. Methods: Vector related pathology and spread after subretinal delivery of AAV2-CBA-RPE65 was studied in RPE65-mutant dogs and normal Spague-Dawley rats. Doses of vector delivered bracketed those intended for the clinical study. Results: There was no systemic toxicity at any vector dose in either species. In dogs ocular examinations showed mild or moderate inflammation that resolved over an initial 3-month period. Retinal histopathology indicated that traumatic lesions from the injection were common, but retinal thinning within the injection region only occurred at the highest vector doses. Biodistribution studies were also performed in RPE65-mutant dogs at various times after vector injection and in normal rats at about 2 weeks and 2 months post-injection. Vector DNA was not widespread outside the injected eye with blood and gonadal tissue consistently negative. Concomitant dose-response results in the RPE65-mutant dogs indicated that the highest 1.5 log unit range of vector doses was efficacious. Conclusions: Given the efficacy and toxicity limits defined in this study, a range for safe vector dose escalation of subretinal AAV2-CBA-RPE65 is suggested for initial human trials.