The aim of this study was to determine the three-dimensional (3D) muscle fascicle architecture in human triceps surae muscles at different contraction levels and muscle lengths. Six male subjects were tested for three contraction levels (0, 30, and 60% of maximal voluntary contraction) and four ankle angles (−15, 0, 15, and 30° of plantar flexion), and the muscles were imaged with B-mode ultrasound coupled to 3D position sensors. 3D fascicle orientations were represented in terms of pennation angle relative to the major axis of the muscle and azimuthal angle (a new architectural parameter introduced in this study representing the radial angle around the major axis). 3D orientations of the fascicles, and the sheets along which they lie, were regionalized in all the three muscles (medial and lateral gastrocnemius and the soleus) and changed significantly with contraction level and ankle angle. Changes in the azimuthal angle were of similar magnitude to the changes in pennation angle. The 3D information was used for an error analysis to determine the errors in predictions of pennation that would occur in purely two-dimensional studies. A comparison was made for assessing pennation in the same plane for different contraction levels, or for adjusting the scanning plane orientation for different contractions: there was no significant difference between the two simulated scanning conditions for the gastrocnemii; however, a significant difference of 4.5° was obtained for the soleus. Correct probe orientation is thus more critical during estimations of pennation for the soleus than the gastrocnemii due to its more complex fascicle arrangement.