Recent recognition of the critical roles of small RNAs in eukaryotic development and metabolism have challenged our conventional thinking about the ways in which genes are regulated in living organisms (Waterhouse et al. 2001a; Carrington and Ambros 2003; Stevenson and Jarvis 2003; Ruvkun et al. 2004). RNA silencing (or gene silencing) is the broad term used to describe mechanisms found in all organisms, with the notable exception of bacteria and the yeast Sacchromyces cerevisiae, variously termed post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. These complex processes involve RNA-RNA, RNA-DNA, RNA-protein, and protein-protein interactions (Wang and Metzlaff 2005). In this chapter, we present an overview of the various RNA silencing pathways, the genes and proteins involved, and the gene silencing technologies that have been developed for RNAi-directed mutagenesis in plants. Most of the key discoveries relating to RNA silencing have been made in the model dicot Arabidopsis. However, the RNA silencing technologies are equally applicable to monocots such as rice. RNA silencing, which has evolved to an extraordinary level of sophistication in the plant kingdom, is a general term used to describe the ensemble of processes involved in virus defense, transposon and chromatin control, and regulation of expression of genes involved in plant development. RNA silencing involves mechanisms that interfere with gene expression by either suppressing gene transcription or initiating sequence-specific mRNA degradation. It can also interfere with gene expression through inhibition of translation, although this occurs less frequently in plants than in animals (Bartel 2004). It has been suggested that these recently discovered gene-silencing mechanisms have several parallels with the immune system of animals (Waterhouse et al. 2001b). © 2007 Springer Science+Business Media, LLC. All rights reserved.