We describe a multistep technique for three-dimensional reconstruction and analysis of brain lesions in vivo, based on the manipulation of magnetic resonance raw data obtained with a special protocol. The technique permits the direct visual identification of neuroanatomical landmarks in each brain specimen and eliminates the need to rely on averaged templates of human brain sections, which can be a source of lesion localization error. The technique also allows for the bidirectional cross-reference between data points in two-dimensional slices and in volume reconstruction and for the projection of subcortical structures onto the three-dimensional cortical surface. The three-dimensional lesion mapping technique can be applied to research with the lesion method in both human and nonhuman primates, to the planning of neurosurgical lesion approach, and to the teaching of neuroanatomy.