RNA interference can induce potent gene silencing through degradation of complementary mRNA. Short double-stranded interfering RNAs are incorporated into an RNA-induced silencing complex that mediates the recognition and degradation of messenger RNAs in a very targeted manner. Though this phenomenon has been described in mammalian cells only a few years ago, there has been an explosion of interest in using small interfering RNAs to efficiently knockdown genes. Consequently, much effort has been put into the development of systems that allow chip and efficient delivery of these molecules into mammalian cells in vitro and in vivo. To overcome the transient inhibitory effects of transfected RNA molecule synthesis in vitro, expression plasmids, mostly based on RNA polymerase III promoters, have been designed to achieve long-term or stable inhibition of the target genes. Moreover, these expression cassettes have been incorporated into viral vectors to obtain gene silencing also in primary cells refractory to plasmid transfection, and to target specific genes in vivo in animal models. The rapid progression in the field of RNA interference has revolutionized the manner in which gene function is studied and, notably, pharmaceutical companies are already validating this technology for medical applications in the near future.