The structure and dynamics of the Dickerson DNA dodecamer [5′d(CGCGAATTCGCG) 2 ] in solution have been investigated by joint simulated annealing refinement against NMR and large-angle X-ray scattering data (extending from 0.25 to 3 Å -1 ). The NMR data comprise an extensive set of heteroand homonuclear residual dipolar coupling and 31 P chemical shift anisotropy restraints in two alignment media, supplemented by NOE and 3 J coupling data. The NMR and X-ray scattering data cannot be fully ascribed to a single structure representation, indicating the presence of anisotropic motions that impact the experimental observables in different ways. Refinement with ensemble sizes (N e ) of g2 to represent the atomic motions reconciles all the experimental data within measurement error. Cross validation against both the dipolar coupling and X-ray scattering data suggests that the optimal ensemble size required to account for the current data is 4. The resulting ensembles permit one to obtain a detailed view of the conformational space sampled by the dodecamer in solution and permit one to analyze fluctuations in helicoidal parameters, sugar puckers, and BI-BII backbone transitions and to obtain quantitative metrics of atomic motion such as generalized order parameters and thermal B factors. The calculated order parameters are in good agreement with experimental order parameters obtained from 13 C relaxation measurements. Although DNA behaves as a relatively rigid rod with a persistence length of ∼150 bp, dynamic conformational heterogeneity at the base pair level is functionally important since it readily permits optimization of intermolecular protein-DNA interactions.