Three-dimensional (3D) or volumetric visualization is a useful resource for learning about the anatomy of the human brain. However, the effectiveness of 3D spatial visualization has not yet been assessed systematically. This report analyzes whether 3D volumetric visualization helps learners to identify and locate subcortical structures more precisely than classical cross-sectional images based on a two dimensional (2D) approach. Eighty participants were assigned to each experimental condition: 2D cross-sectional visualization vs. 3D volumetric visualization. Both groups were matched for age, gender, visual-spatial ability, and previous knowledge of neuroanatomy. Accuracy in identifying brain structures, execution time, and level of confidence in the response were taken as outcome measures. Moreover, interactive effects between the experimental conditions (2D vs. 3D) and factors such as level of competence (novice vs. expert), image modality (morphological and functional), and difficulty of the structures were analyzed. The percentage of correct answers (hit rate) and level of confidence in responses were significantly higher in the 3D visualization condition than in the 2D. In addition, the response time was significantly lower for the 3D visualization condition in comparison with the 2D. The interaction between the experimental condition (2D vs. 3D) and difficulty was significant, and the 3D condition facilitated the location of difficult images more than the 2D condition. 3D volumetric visualization helps to identify brain structures such as the hippocampus and amygdala, more accurately and rapidly than conventional 2D visualization. This paper discusses the implications of these results with regards to the learning process involved in neuroimaging interpretation. Anat Sci Educ. © 2012 American Association of Anatomists.