RNA interference (RNAi) is a process mediated by 21- and 22-nucleotide RNAs (siRNAs) that induce sequence-specific posttranscriptional gene silencing in various organisms. Using a Drosophila in vitro system, the authors demonstrate that siRNAs are generated from long double-stranded RNA (dsRNA) by an RNase III-like processing reaction. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage, and the cleavage site is located near the center of the region spanned by the guiding siRNA. The direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex. The length of dsRNA required for RNAi is between 38 and 501 nucleotides, with shorter dsRNAs being less effective. The 21- and 22-nucleotide fragments generated from dsRNA are unmodified and have 5'-monophosphates and 3'-hydroxyls at their termini, suggesting an RNase III-like mechanism. Synthetic 21- and 22-nucleotide RNAs with overhanging 3' ends mediate target RNA cleavage, and the orientation of the siRNA duplex relative to the protein components determines which strand guides target RNA degradation. The authors propose that the siRNA duplexes are present in a tight ribonucleoprotein complex and do not dissociate during the experiment. The target cleavage site is located near the center of the region covered by the siRNA, and the specificity of target RNA cleavage is exquisite, with no cleavage sites detected outside the region of complementarity to the siRNA.RNA interference (RNAi) is a process mediated by 21- and 22-nucleotide RNAs (siRNAs) that induce sequence-specific posttranscriptional gene silencing in various organisms. Using a Drosophila in vitro system, the authors demonstrate that siRNAs are generated from long double-stranded RNA (dsRNA) by an RNase III-like processing reaction. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage, and the cleavage site is located near the center of the region spanned by the guiding siRNA. The direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex. The length of dsRNA required for RNAi is between 38 and 501 nucleotides, with shorter dsRNAs being less effective. The 21- and 22-nucleotide fragments generated from dsRNA are unmodified and have 5'-monophosphates and 3'-hydroxyls at their termini, suggesting an RNase III-like mechanism. Synthetic 21- and 22-nucleotide RNAs with overhanging 3' ends mediate target RNA cleavage, and the orientation of the siRNA duplex relative to the protein components determines which strand guides target RNA degradation. The authors propose that the siRNA duplexes are present in a tight ribonucleoprotein complex and do not dissociate during the experiment. The target cleavage site is located near the center of the region covered by the siRNA, and the specificity of target RNA cleavage is exquisite, with no cleavage sites detected outside the region of complementarity to the siRNA.