Riboswitches regulate gene expression via specific recognition of cognate metabolites by their aptamer domains, which fold into stable conformations upon ligand binding. However, the recently reported solution and crystal structures of the Bacillus subtilis preQ(1) riboswitch aptamer show small but significant differences, suggesting that there may be conformational heterogeneity in the ligand-bound state. We present a structural and dynamic characterization of this aptamer by solution NMR spectroscopy. The aptamer-preQ(1) complex is intrinsically flexible in solution, with two regions that undergo motions on different time scales. Three residues move in concert on the micro-to-millisecond time scale and may serve as the lid of the preQ(1)-binding pocket. Several Ca2+ ions are present in the crystal structure, one of which binds with an affinity of 47 +/- 2 mu m in solution to a site that is formed only upon ligand binding. Addition of Ca2+ to the aptamer-preQ(1) complex in solution results in conformational changes that account for the differences between the solution and crystal structures. Remarkably, the Ca2+ ions present in the crystal structure, which were proposed to be important for folding and ligand recognition, are not required for either in solution.