RNA interference (RNAi) is a gene silencing mechanism that has been conserved in evolution from yeast to man. Double stranded RNA, which is either expressed by cellular genes for small non-coding RNAs, by parasitic nucleic acids, such as viruses or transposons, or is expressed as an experimental tool, becomes processed into small RNAs, which induce gene silencing by a variety of different means. RNAi-induced gene silencing controls gene expression at all levels, including transcription, mRNA stability and translation. We are only beginning to understand the physiological roles of the RNAi pathway and the function of the many small non-coding RNA species, which are found in eukaryotic genomes. Here we review the known functions of genes in RNAi in various species, the experimental use and design of small RNAs as a genetic tool to dissect the function of mammalian genes and their potential as therapeutic agents to modulate gene expression in patients.