Proteins in thermal equilibrium are associated with conformational distributions rather than single, static structures. Although there are no experimental methods to measure the full protein conformational distribution, several methods exist to probe important aspects. Diffuse X-ray scattering is one such method. We have measured the first three-dimensional reciprocal-space maps of the intensity of diffuse X-ray reflections from protein crystals, and used them to characterize protein conformational distributions. With straightforward modifications, X-ray beamlines can be engineered to enable diffuse scattering measurements for protein crystals. To facilitate future studies, the Lunus software package, used to create the first three-dimensional maps of diffuse X-ray reflections from protein crystals, has been made publicly available ( http://lunus.sourceforge.net ).