Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs^, via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2–R2D2 heterodimer^,, and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery^,,, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2–R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2–R2D2–siRNA complex on Ago2, in a manner dependent on recognition of the 5′-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2–R2D2 and thereby assemble RISC.