Background. Most data on the architecture of the human soleus muscle have been obtained from cadaveric dissection or two-dimensional 2D ultrasound imaging. We present the first comprehensive, quantitative study on the three-dimensional anatomy of the human soleus muscle in vivo using diffusion tensor imaging (DTI) techniques. Methods. We report three-dimensional fascicle lengths, pennation angles, fascicle curvatures, physiological cross-sectional area and volume in four compartments of the soleus at ankle joint angles of 69 ± 12° (plantarflexion, short muscle length; average ± SD across subjects) and 108 ± 7° (dorsiflexion, long muscle length) of six healthy young adults. Microdissection and three-dimensional digitisation on two cadaveric muscles corroborated the compartmentalised structure of the soleus, and confirmed the validity of DTI-based muscle fascicle reconstructions. Results. The posterior compartments of the soleus comprised 80 ± 5% of the total muscle volume (356 ± 58 cm 3). At the short muscle length, the average fascicle length, pennation angle and curvature was 37 ± 8 mm, 31 ±3° and 17 ± 4 /m, respectively. We did not find differences in fascicle lengths between compartments. However, pennation angles were on average 12° larger (p<0.01) in the posterior compartments than in the anterior compartments. For every centimetre that the muscle-tendon unit lengthened, fascicle lengths increased by 3.7 ± 0.8 mm, pennation angles decreased by-3.2 ± 0.9° and curvatures decreased by-2.7 ± 0.8 /m. Fascicles in the posterior compartments rotated almost twice as much as in the anterior compartments during passive lengthening. Discussion. The homogeneity in fascicle lengths and inhomogeneity in pennation angles of the soleus may indicate a functionally different role for the anterior and posterior compartments. The data and techniques presented here demonstrate how DTI can be used to obtain detailed, quantitative measurements of the anatomy of complex skeletal muscles in living humans.