Until recently, only nematodes among animals had a well-defined endogenous small interfering RNA (endo-siRNA) pathway. This has changed dramatically with the recent discovery of diverse intramolecular and intermolecular substrates that generate endo-siRNAs in Drosophila melanogaster and mice. These findings suggest broad and possibly conserved roles for endogenous RNA interference in regulating host-gene expression and transposable element transcripts. They also raise many questions regarding the biogenesis and function of small regulatory RNAs in animals.