The purpose of this study was to establish if there are gender differences in muscle architecture in relaxed human soleus and gastrocnemius muscles of normal, live subjects. Ultrasonography was used to measure fiber bundle length, muscle thickness, and angles of pennation in a total of ten predetermined sites in the medial and lateral heads of gastrocnemius and the anterior and posterior soleus in 19 males and 16 females. Percentage differences between males and females for each parameter were recorded. Gender differences were statistically analyzed using multivariate analysis of variance. In the gastrocnemius and soleus muscles of males and females the differences between the overall fiber bundle length, angle of pennation and muscle thickness were statistically significant (P < 0.05). Overall, females were found to have longer average muscle fiber bundle length and males thicker muscles and larger angles of pennation. The greatest percentage differences of the architectural parameters between males and females were in the posterior soleus: 13% difference in fiber length and 26% difference in angle of pennation in the midline of posterior soleus and 26% difference in muscle thickness of the lateral part of posterior soleus. No correlation was found between leg length and fiber length, angle of pennation or muscle thickness. Fiber length (decreased), angle of pennation (greater) and muscle thickness (greater) of most parts of the gastrocnemius and soleus muscles were significantly different in males and females. Leg length of males and females did not correlate to these architectural parameters.