Five recent publications have documented the successful development and use of gene transfer vectors based on adeno-associated virus (AAV) for expressing short hairpin RNA (shRNA). In cultured mammalian cells and in whole animals, infection with these vectors was shown to result in specific, efficient, and stable knockdown of various targeted endo- or exogenous genes. Here we review this exciting approach, to trigger RNA interference in vitro and in vivo by shRNA expressed from AAV vectors, and describe the state-of-the-art technology for vector particle generation. In particular, we present a set of novel AAV vector plasmids that were specifically designed for the easy and rapid cloning of shRNA expression cassettes into AAV. The plasmids contain alternative RNA polymerase III promoters (U6, H1, or 7SK) together with a respective terminator sequence, as well as stuffer DNA to guarantee an optimal vector size for efficient packaging into AAV capsids. To provide maximum versatility and user-friendliness, the constructs were also engineered to contain a set of unique restriction enzyme recognition sites, allowing the simple and straightforward replacement of the shRNA cassette or other vector components with customized sequences. Our novel vector plasmids complement existing AAV vector technology and should help further establish AAV as a most promising alternative to using adeno- or retro-?lentiviral vectors as shRNA delivery vehicles.