Small interfering RNAs (siRNAs) and Argonaute (Ago) proteins form RNA-induced silencing complexes (RISCs) that silence the expression of target mRNAs. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2/R2D2 heterodimer and the Hsc70/Hsp90 chaperone machinery, the details of RISC assembly remain to be determined. Here, we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsp70, Hsp90, Hop, Hsp40, 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 the recognition of the 5′ phosphate on the siRNA guide strand. We propose that the chaperone machinery acts to support a productive conformation of Ago2, allowing it to load authentic siRNA duplexes from Dicer-2/R2D2. Our results define the molecular basis for the chaperone-assisted assembly of RISC.