PURPOSE: To determine whether the co-injection of extracellular matrix degrading enzymes improves retinal transduction following intravitreal delivery of adeno-associated virus-2 (AAV2). METHODS: AAV2 containing cDNA encoding enhanced green fluorescent protein (GFP), under the control of a chicken β-actin promoter, was delivered by intravitreal injection to adult mice in conjunction with enzymes including collagenase, hyaluronan lyase, heparinase III, or chondroitin ABC lyase. Two weeks later, retinal flatmounts were examined for GFP expression using confocal microscopy. RESULTS: Without the addition of enzymes, transduction was limited to occasional cells in the retinal ganglion cell layer. The addition of heparinase III or chondroitin ABC lyase greatly enhanced transduction of the retinal ganglion cell layer and increased the depth of transduction into the outer retina. Hyaluronan lyase had a limited effect and collagenase was ineffective. Electroretinograms survived with higher concentrations of heparinase III and chondroitin ABC lyase than were required for optimal retinal transduction. CONCLUSIONS: AAV2-mediated retinal transduction is improved by co-injection of heparinase III or chondroitin ABC lyase. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to both the inner and outer retina.