In Drosophila, two features of small interfering RNA (siRNA) structure--5' phosphates and 3' hydroxyls--are reported to be essential for RNA interference (RNAi). Here, we show that as in Drosophila, a 5' phosphate is required for siRNA function in human HeLa cells. In contrast, we find no evidence in flies or humans for a role in RNAi for the siRNA 3' hydroxyl group. Our in vitro data suggest that in both flies and mammals, each siRNA guides endonucleolytic cleavage of the target RNA at a single site. We conclude that the underlying mechanism of RNAi is conserved between flies and mammals and that RNA-dependent RNA polymerases are not required for RNAi in these organisms.