Laser photocoagulation is a well-established treatment modality for retinal disease. Discrete laser burns can be placed anywhere in the retina, singly or multiply, and the burn intensity is controllable. This study investigates the effect of prior laser photocoagulation on the retinal transduction properties of intravitreally administered adeno-associated viral (AAV) vectors. C57BL/6J mice were subjected to unilateral laser photocoagulation 48 hr before bilateral intravitreal injection of self-complementary cytomegaloviral enhanced green fluorescent protein (EGFP) vectors packaged in AAV type 2, 5, and 8 capsids. The eyes were enucleated 4 weeks after injection and examined by histochemistry and quantitative image analysis. Laser pretreatment resulted in substantially increased localized transduction around the burn site for all AAV capsid types. Without laser pretreatment, the vectors transduced only ganglion cells (AAV2) or sporadic cells around the optic nerve head (AAV5 and AAV8). Laser pretreatment increased AAV2 vector expression throughout the entire retina and focally at the burn site. Transduced cells at the burn site included retinal pigment epithelium (RPE), photoreceptors, Müller cells, inner nuclear layer cells, and retinal ganglion cells. The AAV5 vector showed increased RPE transduction at the burn site only. The AAV8 vector showed augmented expression in RPE, photoreceptors, and Müller cells around the burn site. Migrating RPE cells, present in the neural retina near the burn site, were also transduced by all three capsid types as evidenced by colocalization of EGFP and cytokeratin. Laser photocoagulation can be used to precisely direct AAV vector transduction to discrete locations in the retina. A combination of laser and AAV-mediated gene expression may allow the development of improved therapies for diabetic retinopathy, branch and central vein occlusion, and age-related macular degeneration.