Ultrasonography was used to measure the pennation angle of the human tibialis anterior (TA), lateral gastrocnemius (LG), medial gastrocnemius (MG), and soleus (Sol). The right and left legs of 8 male and 8 female subjects were tested at rest and during maximum voluntary contraction (MVC). Joint angles were chosen to control muscle tendon lengths so that the muscles were near their optimal length within the length– tension relationship. No differences in pennation angle were detected between the right and left legs. Another consistent finding was that the pennation angle at MVC was significantly greater than at rest for all muscles tested. Optimal pennation angles for the TA, MG, and Sol were significantly greater for the men than for the women. Optimal pennation angles for the TA, LG, MG, and Sol for the male subjects were 14.3°, 23.7°, 34.6°, and 40.1° respectively, whereas values of 12.1°, 16.3°, 27.3°, and 26.3° were recorded for the female subjects. The results of this study suggest the following: (1) similar values for pennation angle can be used for the right and left TA, LG, MG, and Sol; (2) pennation angle is significantly greater at MVC than at rest for all muscles tested; and (3) sex-specific values for optimal pennation angle should be used when modeling the force-generating potential of the primary muscles responsible for ankle plantar and dorsiflexion.