In bacteria, small anti-sense RNAs (sRNAs) help control the cell's response to environmental stress by binding complementary sequences in their mRNA targets. Hfq protein is required for the regulatory function of anti-sense sRNAs, and facilitates the formation of sRNA-mRNA complexes. Hfq is a ring-shaped homohexamer that preferentially binds single-stranded U-rich RNA on its proximal face and A-rich RNA on its distal face. Although Hfq has been shown to accelerate RNA base pairing and strand exchange in vitro, how Hfq does this is still unknown. To model the annealing mechanism of Hfq, we used a U-rich16-nt RNA (D16). Stopped-flow FRET measurements using fluorescein-labeled D16 RNA and Cy3-labeled Hfq showed that Hfq binds single-stranded RNA rapidly (≥ 7•107M−1s−1). Upon the addition of a complementary RNA, Hfq is rapidly released from the RNA complex (kobs ∼ 30 s−1). Stopped-flow experiments with a fluorescent molecular beacon showed that Hfq accelerates RNA annealing and strand exchange one hundred times. However, annealing is slower than Hfq release, suggesting the protein dissociates before the duplex is fully formed. Hfq mutations and competition experiments suggest that D16 RNA and the RNA beacon interact with the proximal and distal faces of Hfq, respectively, as expected. When a DNA beacon that binds Hfq weakly (1.4 μM) is used instead of an RNA beacon, Hfq-mediated RNA annealing is faster, but saturates at a higher concentration of Hfq. These results are consistent with rapid binding of RNA to the distal and proximal faces of Hfq, forming a transient ternary complex. We propose that Hfq leaves the complex before RNA hybridization is complete, after which the RNA strands form a stable duplex or dissociate and rebind Hfq as single-strands for the next round of annealing.