Diffusion tensor (DT) MRI provides the directional information of water molecular diffusion, which can be utilized to estimate the connectivity of white matter tract pathways in the human brain. Several white matter tractography methods have been developed to reconstruct the white matter fiber tracts using DT-MRI. With conventional methods (e.g., streamline techniques), however, it would be very difficult to trace the white matter tracts passing through the fiber crossing and branching regions due to the ambiguous directional information with the partial volume effect. The purpose of this study was to develop a new white matter tractography method which permits fiber tract branching and passing through crossing regions. Our tractography method is based on a three-dimensional (3D) directional diffusion function (DDF), which was defined by three eigenvalues and their corresponding eigenvectors of DT in each voxel. The DDF-based tractography (DDFT) consists of the segmentation of white matter tract region and fiber tracking process. The white matter tract regions were segmented by thresholding the 3D directional diffusion field, which was generated by the DDF. In fiber tracking, the DDFT method estimated the local tract direction based on overlap of the DDFs instead of the principal eigenvector, which has been used in conventional methods, and reconstructed tract branching by means of a one-to-many relation model. To investigate the feasibility and usefulness of the DDFT method, we applied it to DT-MRI data of five normal subjects and seven patients with a brain tumor. With the DDFT method, the detailed anatomy of white matter tracts was depicted more appropriately than the conventional methods.