Diffusion-tensor magnetic resonance imaging (DT-MRI) offers great potential for understanding structure-function relationships in human skeletal muscles. The purposes of this study were to demonstrate the feasibility of using in vivo human DT-MRI fiber tracking data for making pennation angle measurements and to test the hypothesis that heterogeneity in the orientation of the tibialis anterior (TA) muscle's aponeurosis would lead to heterogeneity in pennation angle. Eight healthy subjects (5 male) were studied. T1-weighted anatomical MRI and DT-MRI data were acquired of the TA muscle. Fibers were tracked from the TA's aponeurosis by following the principal eigenvector. The orientations of the aponeurosis and muscle fiber tracts in the laboratory frame of reference and the orientation of the fiber tracts with respect to the aponeurosis [i.e., the pennation angle (θ)] were determined. The muscle fiber orientations, when expressed relative to the laboratory frame of reference, did not change as functions of superior-to-inferior position. The sagittal and coronal orientations of the aponeurosis did not change in practically significant manners either, but the aponeurosis′ axial orientation changed by ∼40°. As a result, the mean value for θ decreased from 16.3 (SD 6.9) to 11.4° (SD 5.0) along the muscle's superior-to-inferior direction. The mean value of θ was greater in the deep than in the superficial compartment. We conclude that pennation angle measurements of human muscle made using DT-MRI muscle fiber tracking are feasible and reveal that in the foot-head direction, there is heterogeneity in the pennation properties of the human TA muscle.