One of the obstacles to efficient vector-mediated gene transfer to the CNS is limited transduction of target neurons. The VP22 tegument protein of HSV-1 can cross biological membranes and translocate the VP22 protein from primarily transfected cells to many surrounding cells in vitro. Here, we employed an adenoviral vector coding for a VP22-GFP fusion protein driven by a CMV promoter to test its capability of transducing CNS neurons in vivo. Intraocular administration of Ad.VP22-GFP in the rat doubled both the retinal area containing transduced, GFP-expressing cells and the absolute number of GFP-expressing retinal neurons compared to Ad.GFP transduction. Following injection of Ad.VP22-GFP into the mouse brain, the transduced striatal area was increased by a factor of 7 compared to intracerebral injection of Ad.GFP. In both retina and striatum, GFP-expressing cells were identified as mainly neurons. Thus, VP22 greatly augments adenovirus-mediated transgene delivery to CNS neuronsin vivo, making VP22 a promising tool for enhancing the efficacy of adenoviral gene transfer of protective factors to the CNS.