RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.