The Mouse Biomedical Informatics Research Network (MBIRN) has been established to integrate imaging studies of the mouse brain ranging from three-dimensional (3D) studies of the whole brain to focused regions at a sub-cellular scale. Magnetic resonance (MR) histology provides the entry point for many morphologic comparisons of the whole brain. We describe a standardized protocol that allows acquisition of 3D MR histology (43-microm resolution) images of the fixed, stained mouse brain with acquisition times <30 min. A higher resolution protocol with isotropic spatial resolution of 21.5 microm can be executed in 2 h. A third acquisition protocol provides an alternative image contrast (at 43-microm isotropic resolution), which is exploited in a statistically driven algorithm that segments 33 of the most critical structures in the brain. The entire process, from specimen perfusion, fixation and staining, image acquisition and reconstruction, post-processing, segmentation, archiving, and analysis, is integrated through a structured workflow. This yields a searchable database for archive and query of the very large (1.2 GB) images acquired with this standardized protocol. These methods have been applied to a collection of both male and female adult murine brains ranging over 4 strains and 6 neurologic knockout models. These collection and acquisition methods are now available to the neuroscience community as a standard web-deliverable service.