Small RNAs guide RNA-induced silencing complexes (RISCs) to bind to cognate mRNA transcripts and trigger silencing of protein expression during RNA interference (RNAi) in eukaryotes. A fundamental aspect of this process is the asymmetric loading of one strand of a short interfering RNA (siRNA) or microRNA (miRNA) duplex onto RISCs for correct target recognition. Here, we use a reconstituted system to determine the extent to which the core components of the human RNAi machinery contribute to RNA guide strand selection. We show that Argonaute2 (Ago2), the endonuclease that binds directly to siRNAs and miRNAs within RISC, has intrinsic but substrate-dependent RNA strand selection capability. This activity can be enhanced substantially when Ago2 is in complex with the endonuclease Dicer and the double-stranded RNA-binding proteins (dsRBPs)-trans-activation response (TAR) RNA-binding protein (TRBP) or protein activator of PKR (PACT). The extent to which human Dicer/dsRBP complexes contribute to strand selection is dictated by specific duplex parameters such as thermodynamics, 5' nucleotide identity, and structure. Surprisingly, our results also suggest that strand selection for some miRNAs is enhanced by PACT-containing complexes but not by those containing TRBP. Furthermore, overall mRNA targeting by miRNAs is disfavored for complexes containing TRBP but not PACT. These findings demonstrate that multiple proteins collaborate to ensure optimal strand selection in humans and reveal the possibility of delineating RNAi pathways based on the presence of TRBP or PACT.