RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS) [1]. In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21–23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation [2]. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types [3–7], whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells [8]. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS [9–11], little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1–4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.