Diffusion tensor magnetic resonance imaging (MRI) was used to estimate white matter fiber orientations in fixed brain specimens. The specimens were subsequently sectioned, stained for myelinated fibers, and imaged with light microscopy. The MRI data were registered with the micrographs, allowing direct comparison of fiber orientation estimates between the two methods. Fiber orientation was measured in regions of interest in the frontal lateral corpus callosum. The results of diffusion MRI and light microscopy agreed within 2 degrees-the difference was not statistically significant.