We used diffusion tensor imaging to assess diffusion anisotropy in the pyramidal tract in ten young, and ten elderly subjects (five males and five females in each group). The purpose of this study was to define normative values for anisotropy at different anatomic levels of the brainstem as well as to assess differences due to age, gender, and laterality. In all subjects, anisotropy was highest in the cerebral peduncle, lowest in the caudal pons, and intermediate in the medulla. In the pons and medulla the regional variability was high, with significant differences in anisotropy even between contiguous slices. Multifactorial ANOVA (performed using the average value of anisotropy within each region of interest) revealed that elderly subjects had significantly lower values than young subjects in the cerebral peduncle, with no differences in the pons and medulla. No significant differences in anisotropy due to gender and side were found. The differences in anisotropy at different levels of the brainstem reflect differences in the local architecture of white matter fibers. Anisotropy is high in the cerebral peduncle because fibers have a highly ordered arrangement, while in the pons and medulla, anisotropy is lower because the local fiber architecture is less coherent due to the presence of other fibers and nuclei. The biologic meaning of the intergroup differences in anisotropy is discussed in light of the structure and architecture of the tissue under investigation. We also consider potential sources of artifacts, such as noise and motion, partial volume contamination, anatomic mismatching, and the use of inappropriate statistical tests. We conclude that the age-related decrease in anisotropy in the cerebral peduncle is not artifactual but rather reflects subtle structural changes of the aging white matter. Our study however shows that caution must be exercised in interpreting diffusion anisotropy data.