Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system. Display omitted •Binding energies, association, and cleavage rates measured for >40,000 RISC targets•AGO2 tolerates large insertions in the target opposite the central region of the guide•Some guide:target mismatches enhance the single-turnover RISC cleavage rate•In vitro measured biochemical parameters explain knockdown in cells By high-throughput, quantitative characterization of binding and cleavage for >40,000 distinct RISC targets, Becker et al. reveal principles of miRNA regulation and siRNA function. These data enable construction of quantitative models of binding and cleavage and are used to explain mRNA knockdown in cells.