Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm(2). AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS.