The position and extent of individual fiber tracts within the white matter of human brains can be identified in vivo using diffusion tensor imaging (DTI) and fiber tracking methods. Previous to this study, however, the lack of three-dimensional (3-D) probability maps precluded comparing the anatomical precision of MRI studies with microscopically defined fiber tracts in human postmortem brains. The present study provides 3-D registered maps of the topography, course and intersubject variability of major fiber tracts, which were identified at microscopic resolution. The analyzed tracts include the corticospinal tract, optic and acoustic radiations, fornix, cingulum, corpus callosum, superior longitudinal, superior and inferior occipito-frontal and uncinate fascicles; sources and targets of fiber tracts include the lateral and medial geniculate nuclei and mamillary bodies. Tracts and nuclei were identified in serial myelin-stained histological sections of ten postmortem brains. The sections were 3-D reconstructed and registered to a standardized stereotaxic space of an in vivo MR reference brain by means of linear and non-linear, elastic transformations. The individual fiber tracts and nuclei were superimposed in the reference space, and probability maps were generated as a quantitative measure of intersubject variability for each voxel of the stereotaxic space. This study presents the first stereotaxic atlas of the course, location and extent of fiber tracts and related nuclei based on microscopically defined localization and topographic data taken at multiple levels on each of the three orthogonal planes. The maps are useful for evaluating and identifying fiber bundles in DTI, for localizing subcortical lesions visible in anatomical MR images and for studying neuronal connectivity.