DEAD-box RNA helicases bind and remodel RNA and RNA-protein complexes in an ATP-dependent fashion. Several lines of evidence suggest that DEAD-box RNA helicases can also form stable, persistent complexes with RNA in a process referred to as RNA clamping. The molecular basis of RNA clamping is not well understood. Here we show that the yeast DEAD-box helicase Ded1p forms exceptionally long-lived complexes with RNA and the nonhydrolyzable ATP ground-state analogue ADP-BeFx or the nonhydrolyzable ATP transition state analogue ADP-AlFx. The complexes have lifetimes of several hours, and neither nucleotide nor Mg(2+) is released during this period. Mutation of arginine 489, which stabilizes the transition state, prevents formation of long-lived complexes with the ATP transition state analogue, but not with the ground state analogue. We also show that two other yeast DEAD-box helicases, Mss116p and Sub2p, form comparably long-lived complexes with RNA and ADP-BeFx. Like Ded1p, Mss116p forms long-lived complexes with ADP-AlFx, but Sub2p does not. These data suggest that the ATP transition state might vary for distinct DEAD-box helicases, or that the transition state triggers differing RNA binding properties in these proteins. In the ATP ground state, however, all tested DEAD-box helicases establish a persistent grip on RNA, revealing an inherent capacity of the enzymes to function as potent, ATP-dependent RNA clamps.