Precisely measuring the ensemble of conformers that a macromolecule populates in solution is highly challenging. Thus, it has been difficult to confirm or falsify the predictions of nanometer-scale dynamical modeling. Here, we apply an X-ray interferometry technique to probe the solution structure and fluctuations of B-form DNA on a length scale comparable to a protein-binding site. We determine an extensive set of intrahelix distance distributions between pairs of probes placed at distinct points on the surface of the DNA duplex. The distributions of measured distances reveal the nature and extent of the thermally driven mechanical deformations of the helix. We describe these deformations in terms of elastic constants, as is common for DNA and other polymers. The average solution structure and microscopic elasticity measured by X-ray interferometry are in striking agreement with values derived from DNA-protein crystal structures and measured by force spectroscopy, with one exception. The observed microscopic torsional rigidity of DNA is much lower than is measured by single-molecule twisting experiments, suggesting that torsional rigidity increases when DNA is stretched. Looking forward, molecular-level interferometry can provide a general tool for characterizing solution-phase structural ensembles.Au-SAXS | bending rigidity | twisting rigidity | persistence length | bases per helical turn A central lesson from the last 40 y of structural biology is that proteins and nucleic acids populate multiple conformational states in solution and that transitions between the states produce biological function. Despite the importance of such conformational fluctuations, there is a dearth of tools to quantitatively measure the ensemble of conformers that is present in solution. NMR structures are often reported as ensembles, but these ensembles represent a combination of actual molecular flexibility and experimental uncertainty. More recently, conformationalaveraged order parameters derived from residual dipolar coupling data have been used to parameterize ensemble models (1, 2). These models call for testing by an independent experimental measure.The distances between points in a macromolecule are closely related to the 3D structure of the macromolecule. This close relationship is because interpoint distances determine the relative position of the points in space in a model-free way (allowing for global rotation, translation, or reflection). For a macromolecule with a dynamic conformation, distance distributions between many different pairs of points, in conjunction with a multibody or elastic model, can define the macromolecule's structural ensemble.Thus, in principle, molecular rulers provide the required experimental information: intramolecular distance distributions. However, whereas existing rulers are sensitive reporters of ordinal change in intramolecular distance, they do not give absolute distances or accurate occupancy distributions when multiple distinct distances (conformations) coexist. These limitations arise...