The purpose of our study was to evaluate the biodistribution of rAAV vectors following subretinal or intravitreal injection. In rats, we performed subretinal or intravitreal injections of rAAV-2/2.CMV.gfp. In large animals, rAAV-2/4.CMV.gfp or rAAV-2/5.CMV.gfp was delivered into the subretinal space while rAAV-2/2.CMV.gfp was delivered either to the subretinal space or to the vitreous. In euthanized animals, we undertook a complete necropsy. In animals maintained alive, we collected blood and tissue samples from the submandibular lymph node, liver, and gonads. We analyzed total DNA, extracted from various tissue samples and peripheral blood mononuclear cells (PBMC), by PCR. Following subretinal or intravitreal injections in rats and in large animals, vector sequences were not detected in the liver or in the gonads but were occasionally found in PBMC. An unexpected result was the detection of rAAV sequences in the optic nerve following subretinal injection. The most striking finding was the detection of vector sequences in the brain, along the visual pathway, in rAAV-2/2 intravitreally injected dogs. These findings raise safety concerns regarding intraocular administration of rAAV vectors and will have an impact on the development of future gene therapy trials for retinal diseases.