The functions of most RNA molecules are critically dependent on the distinct local dynamics that characterize secondary structure and tertiary interactions and on structural changes that occur upon binding by proteins and small molecule ligands. Measurements of RNA dynamics at nucleotide resolution set the foundation for understanding the roles of individual residues in folding, catalysis, and ligand recognition. In favorable cases, local order in small RNAs can be quantitatively analyzed by NMR in terms of a generalized order parameter, S2. Alternatively, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemistry measures local nucleotide flexibility in RNAs of any size using structure-sensitive reagents that acylate the 2′-hydroxyl position. In this work, we compare per-residue RNA dynamics, analyzed by both S2 and SHAPE, for three RNAs: the HIV-1 TAR element, the U1A protein binding site, and the Tetrahymena telomerase stem loop 4. We find a very strong correlation between the two measurements: nucleotides with high SHAPE reactivities consistently have low S2 values. We conclude that SHAPE chemistry quantitatively reports local nucleotide dynamics and can be used with confidence to analyze dynamics in large RNAs, RNA-protein complexes, and RNAs in vivo. Copyright © 2008 American Chemical Society.